Targeted cleavage of RNA using ribonuclease P targeting and cleavage sequences

ABSTRACT

It has been discovered that any RNA can be targeted for cleavage by RNase P from prokaryotic or eukaryotic cells using a suitably designed oligonucleotide (&#34;external guide sequence&#34;, or EGS) to form a hybrid with the target RNA, thereby creating a substrate for cleavage by RNase P in vitro. The EGS hydrogen bonds to the targeted RNA to form a partial tRNA like structure including the aminoacyl acceptor stem, the T stem and loop, and part of the D stem. An EGS can be modified both by changes in sequence and by chemical modifications to the nucleotides. The EGS can be a separate molecule or can be combined with an RNase P catalytic RNA sequence to form a single oligonucleotide molecule (&#34;RNase P internal guide sequence&#34; or RIGS). Methods are also disclosed to randomly select and to express a suitable EGS or RIGS in vivo to make a selected RNA a target for cleavage by a host cell RNase P or introduced RIGS, thus preventing expression of the function of the target RNA. The methods and compositions should be useful to prevent the expression of disease- or disorder-causing genes in vivo.

This application is a continuation-in-part of U.S. Ser. No. 08/207,547,entitled "Targeted Cleavage of RNA Using Eukaryotic Ribonuclease P andExternal Guide Sequence" filed Mar. 7, 1994, and a continuation-in-partof U.S. Ser. No. 08/215,082, entitled "Targeted Cleavage of RNA UsingEukaryotic Ribonuclease P and External Guide Sequence" filed Mar. 18,1994. This is a national stage application under 35 U.S.C. § 371 ofPCT/US95/02816.

BACKGROUND OF THE INVENTION

This invention is in the general area of genetic engineering of nucleicacid sequences, especially chemically modified external guide sequencesand catalytic RNA sequences linked to guide sequences.

There are several classes of ribozymes now known which are involved inthe cleavage and/or ligation of RNA chains. A ribozyme is defined as anenzyme which is made of RNA, most of which work on RNA substrates.Ribozymes have been known since 1982, when Cech and colleagues (Cell 31:147-157) showed that a ribosomal RNA precursor in Tetrahymena, aunicellular eukaryote, undergoes cleavage catalyzed by elements in theRNA sequence to be removed during the conversion of the rRNA precursorinto mature rRNA. Another class of ribozyme, discovered in 1983, was thefirst to be shown to work in trans, that is, to work under conditionswhere the ribozyme is built into one RNA chain while the substrate to becleaved is a second, separate RNA chain. This ribozyme, called M1 RNA,was characterized in 1983 by Altman and colleagues as responsible forthe cleavage which forms mature 5' ends of all transfer RNAs (tRNAs) inE. coli. Analogous RNA-containing enzymes concerned with tRNA synthesishave since been found in all cells in which they have been sought,including a number of human cell lines, though the relevant eukaryoticRNAs have not yet been shown to be catalytic by themselves in vitro.

The discovery and characterization of this catalytic RNA is reviewed bySidney Altman, in "Ribonuclease P: An Enzyme with a Catalytic RNASubunit" in Adv. Enzymol. 62: 1-36 (1989). The activity was firstisolated from E. coli extracts, and subsequently determined to be aribonucleoprotein having two components, an RNA component called M1 anda protein component called C5. The RNA cleaved substrates in a trueenzymatic reaction, as measured using Michaelis-Menten kinetics. M1 wasdetermined to be solely responsible for substrate recognition and C5 wasdetermined to alter k_(cat) but not K_(M), as reported byGuerrier-Takada et al., Cell 35: 849 (1983) and McClain et al., Science238: 527 (1987). Sequencing showed that M1 RNA is 377 nucleotides long,M_(r) approximately 125,000, and that the protein consists of 119 aminoacids, M_(r) approximately 13,800, as reported by Hansen et al., Gene38: 535 (1987).

Cleavage of precursor tRNA molecules by the RNA component of eubacterialRNase P is described by Guerrier-Takada et al., Cell 35, 849 (1983) andreviewed by Altman, Adv. Enzymol. 62:1 (1989).

U.S. Pat. No. 5,168,053 entitled "Cleavage Of Targeted RNA By RNase P"to Altman et al., discloses that it is possible to target any RNAmolecule for cleavage by bacterial RNase P by forming a nucleotidesequence part of which is complementary to a targeted site and whichincludes a terminal 3'-NCCA, wherein the sequence is designed tohybridize to the targeted RNA so that the bacterial RNase P cleaves thesubstrate at the hybrid base-paired region. Specificity is determined bythe complementary sequence. The sequence is preferably ten to fifteennucleotides in length and may contain non-complementary nucleotides tothe extent this does not interfere with formation of several base pairsby the complementary sequence which is followed by NCCA at the 3' end.

As described in WO 92/03566 to Yale University, ribonuclease P (RNase P)from E. coli can cleave oligoribonucleotides that are found inhydrogen-bonded complexes that resemble the aminoacyl stem and includethe 5' leader sequence of tRNA precursors, --NCAA. Human RNase P cannotcleave in vitro the 5' proximal oligoribonucleotide in the simplecomplexes cleaved by RNase P from E. coli, but can do so when the 3'proximal oligoribonucleotide is bound to an external guide sequence(EGS) to form a structure resembling portions of a tRNA molecule. TheEGS can include a complementary sequence to a target substrate of atleast eleven nucleotides, seven bases which hydrogen bond to thetargeted sequence to form a structure akin to the aminoacyl acceptorstem of a precursor tRNA, and four nucleotides which base pair with thetargeted sequence to form a structure akin to the dihydroxyuracil stem.WO 92/03566 does not disclose EGS for prokaryotic RNase P with fewerthan seven complementary nucleotides.

WO 93/22434 to Yale University discloses an EGS for human RNase P. Asdescribed in WO 93/22434, an EGS for human RNase P consists of asequence which, when in a complex with the target substrate molecule,forms a secondary structure resembling that of a tRNA cloverleaf, or asubstantial part of it, and that results in cleavage of the 10 targetRNA by RNase P. The sequence of the EGS of WO 93/22434 is derived fromany tRNA except that the D stem and aminoacyl stem are altered to becomplementary to the target substrate sequence. WO 93/22434 alsodiscloses EGS with either the anticodon loop and stem or the extra loopdeleted, and EGS where the sequence of the T loop and stem are changed.WO 93/22434 does not disclose eukaryotic EGS comprising only a regioncomplementary to the target RNA and a region forming a structure similarto only the T stem and loop of tRNA. Neither WO 92/03566 nor WO 93/22434discloses EGS having chemically modified nucleotides.

It is therefore an object of the present invention to provide methodsand compositions for specifically cleaving targeted RNA sequences usinglinked catalytic RNA and minimal guide sequences.

It is another object of the present invention to provide chemicallymodified external guide sequences for RNase P with enhanced resistanceto nuclease degradation.

It is another object of the present invention to provide a method forselecting external guide sequences, and linked catalytic RNA and guidesequences, that cleave a target RNA with increased efficiency.

It is a further object of the present invention to provide methods andcompositions for specifically cleaving RNA, both in vitro and in vivowithin eukaryotic cells, for the treatment of disease conditions whichinvolve RNA transcription or translation, such as diseases caused by RNAand DNA viruses and expression of excessive or pathogenic proteins frommRNA, or of excessive or pathogenic RNA, itself.

SUMMARY OF THE INVENTION

Any RNA can be targeted for cleavage by RNase P, using a suitablydesigned oligonucleotide ("external guide sequence") to form a hybridwith the target RNA, thereby creating a substrate targeted for cleavageby RNase P. The EGSs contain sequences which are complementary to thetarget RNA and which forms secondary and tertiary structure akin toportions of a tRNA molecule. A eukaryotic EGS must contain at leastseven nucleotides which base pair with the target sequence 3' to theintended cleavage site to form a structure like the amino acyl acceptorstem, nucleotides which base pair to form a stem and loop structuresimilar to the T stem and loop, followed by at least three nucleotidesthat base pair with the target sequence to form a structure like thedihydroxyuracil stem. The EGS can be made more resistant to nucleasedegradation by including chemically modified nucleotides or nucleotidelinkages.

The external guide sequence and the RNase P catalytic RNA can be usedtogether as separate molecules. Alternatively, the two sequences can becombined into a single oligonucleotide molecule possessing bothtargeting and catalytic functions. Such a combined oligonucleotide,termed an RNase P internal guide sequence (RIGS), increases the kineticefficiency of cleavage by reducing the number of reactants and bykeeping the targeting and catalytic elements in close proximity.Chemically modifying the nucleotides and phosphate linkages of EGSmolecules and RIGS molecules make the oligonucleotides more resistant tonuclease degradation.

Methods are also disclosed to select RIGS molecules and EGS moleculeshaving increased substrate affinity and utility in vivo to cleave ortarget cleavage of a selected RNA, thus preventing expression of thefunction of the target RNA. The methods and compositions should beuseful to prevent the expression of disease- or disorder-causing genesin vivo.

As described in the examples, an RIGS was constructed by linking a guidesequence to M1 RNA (M1GS RNA). M1GS RNA can act as a sequence-specificendonuclease and can cleave target RNAs that base pair with the guidesequence just as group I introns do. A custom-designed M1GS RNA cleavesthe mRNA that encodes thymidine kinase (TK) of human herpes simplexvirus 1 (HSV-1) in vitro. When this M1GS RNA is expressed in mammaliancells in tissue culture, it reduces the level of expression of TK bydecreasing the amount of the target TK mRNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the proposed secondary structure of the chimeric substrateused in the selection procedure (Sequence ID No. 1). The italicizedsequence is from CAT mRNA and the remaining sequence, aside from changesmade to assure hydrogen bonding specifically to CAT mRNA, is based onthe sequence of E. coli tRNA^(Tyr). The nine nucleotides that wererandomized are each indicated by N. Some of the EGS analogs for variousparts of a tRNA are also depicted: acceptor stem, T loop, anticodonloop, and D-loop.

FIG. 2 depicts the general scheme for in vitro selection andamplification of chimeric substrates with enhanced efficiency fordirecting human RNase P cleavage. "Reverse transcriptase" denotes thatthe DNA polymerase capability of reverse transcriptase was used tocreate double-stranded template DNA from the overlapping DNAoligonucleotides SEC-1A (Sequence ID No. 2) and SEC-1B (Sequence ID No.3). "RNA-PCR" refers to reverse transcription coupled to PCR.

FIG. 3 is the sequence and secondary structures of the precursor totRNA^(Tyr) (Sequence ID No. 4) and a complex of a substrate andEGSΔ(1-18) (Sequence ID No. 5), which is derived from tRNA^(Tyr) butlacks the first eighteen nucleotides from the 5' end of the maturetRNA^(Tyr). pTyr: E. coli tRNA^(Tyr) precursor; pAva: substrate (target)RNA with 5' leader sequence and the first fourteen nucleotides of E.coli tRNA^(Tyr) (Sequence ID No. 6).

FIGS. 4A, 4B, 4C, 4D, 4E, and 4F are the proposed secondary structuresof complexes of CAT mRNA and various EGS. 4A and 4D depict complexesbetween the complementary region CAT mRNA (Sequence ID No. 7) andEGS^(CAT) (Sequence ID No. 8). 4B and 4E are complexes of CAT mRNA(Sequence ID No. 7) and EGS 9 (Sequence ID No. 9). 4C and 4F arecomplexes of CAT mRNA (Sequence ID No. 7) and EGS^(CAT) ΔAC (Sequence IDNo. 10). Hollow arrows denote sites of cleavage by human RNase P. InFIGS. 4A, 4B and 4C solid arrowheads denote the sites of cleavage byRNase T1, and arrows denote sites of cleavage by RNase T2. In FIGS. 4D,4E and 4F, sites of cleavage by cobra venom nuclease are indicated bysolid arrows.

FIG. 5 shows rates (nmol/min) of human RNase P cleavage of CAT mRNAdirected by twelve individual EGS RNAs. Nine individual EGS RNAs: EGS 1(Sequence ID No. 17), EGS 4 (Sequence ID No. 19), EGS 5 (Sequence ID No.20), EGS 6 (Sequence ID No. 21), EGS 8 (Sequence ID No. 22), EGS 9(Sequence ID No. 9), EGS 12 (Sequence ID No. 26), EGS 14 (Sequence IDNo. 27), EGS 18 (Sequence ID No. 31), were prepared by in vitroselection. EGS 19 and EGS^(CAT) ΔAC (Sequence ID No. 10) were preparedby in vitro mutagenesis. Results are presented as initial rates(nmol/mol) of cleavage of substrate by RNase P during the linear phaseof each reaction.

FIGS. 6A, 6B, and 6C are sequence and proposed secondary structures ofEGS for herpes simplex virus thymidine kinase mRNA (Sequence ID No. 35).FIG. 6A is an EGS (Sequence ID No. 36) forming an aminoacyl acceptorstem, T loop and stem, variable loop and stem, anticodon loop and stem,and D stem. FIG. 6B is an EGS (Sequence ID No. 37) wherein a G in the Tloop is substituted for a C in the EGS of FIG. 6A. FIG. 6C is an EGS(Sequence ID No. 38) where the anticodon loop and stem of the EGS ofFIG. 6A is deleted.

FIG. 7 is a graph of percent TK mRNA expression for cell lines: CL-CAT,CL-109, CL-104, and CL-112.

FIG. 8 is the sequence and structure of H1 RNA (Sequence ID No. 11) fromAltman et al., Genomics 18: 418-422 (1993).

FIG. 9 is a schematic representation of complexes that are formed by M1GS RNA (Sequence ID No. 51 and Sequence ID No. 52) and two substrates(Sequence ID No. 53 and Sequence ID No. 54).

FIG. 10 is a diagram of TK and CaT RNA substrate RNAs. The two 13-ntsegments highlighted in the TK RNA and CAT RNA sequences (Sequence IDNo. 55 and Sequence ID No. 56) are complementary to the guide sequencesin M1TK13 and M1CAT13 RNAs, respectively. The cleavage sites areindicated by arrows.

FIG. 11 is a schematic representation of the tk46 substrate sequence(Sequence ID No. 57) and the structure of RNA enzymes used (Sequence IDNo. 58, Sequence ID No. 59, Sequence ID No. 60, Sequence ID No. 61,Sequence ID No. 62, Sequence ID No. 63, Sequence ID No. 64, and SequenceID No. 65). The arrow marks the expected site of cleavage. The sequencesshown in the M1GS RNAs! bold type are the guide sequences that containthe 3' CCA sequence and the sequence complementary to the TK sequence.

FIG. 12 is a schematic representation of retroviral vectors (ΔM1TK,M1TK, and NB2) containing M1GS RNAs or an EGS sequence complementary tothe influenza viral protein PB2.

FIG. 13 is a graph of levels of expression (percent of controlexpression) of TK mRNA and protein in ψCRE cells and in other celllines. Values are averages of results from four independent experiments.Results from the four experiments varied within 5% in absolute terms.

Solid bars: TK mRNA; open bars; TK protein.

DETAILED DESCRIPTION OF THE INVENTION Guide Sequence

As used herein, a guide sequence (GS) is an oligonucleotide that targetsa substrate RNA molecule for cleavage by a catalytic RNA having theactivity of RNase P catalytic RNA. A guide sequence may be a separatemolecule, termed an external guide sequence, or combined in a singlemolecule with catalytic RNA. Such a combined molecule is referred toherein as an RNase P internal guide sequence (RIGS).

A. Eukaryotic RNase P Targeting Sequence

A guide sequence for human RNase P consists of a sequence which, when ina complex with the target substrate molecule, forms a secondarystructure resembling that of a tRNA cloverleaf or a part of it.

As used herein, the term "resembling a precursor tRNA" means a complexformed by the GS with target RNA substrate to resemble a sufficientportion of the tRNA secondary and tertiary structure to result incleavage of the target RNA by RNase P. The sequence of the GS can bederived from any tRNA except that the D stem and aminoacyl stem have tobe altered to be complementary to the target substrate sequence. Thesealtered stems are referred to as recognition arms. The recognition armcorresponding to the aminoacyl stem is referred to as the A recognitionarm and the recognition arm corresponding to the D stem is referred toas the D recognition arm. The remaining portion of the guide sequence,which is required to cause RNase P catalytic RNA to interact with theGS/target sequence complex, is herein referred to as RNase P bindingsequence. The presence of a 3'-CCA on an EGS enhances the efficiency ofin vitro reaction with the human RNase P by about 35%. The anticodonloop and stem and extra loop can separately be deleted and the sequenceof the T loop and stem can be changed without decreasing the usefulnessof the guide sequence and, in the case of the anticodon stem and loopdeletion, increases the efficiency of the reaction by about ten fold.Changes in other parts of an EGS can increase its efficiency about onehundred fold.

The desired secondary structure is determined using conventionalWatson-Crick base pairing schemes to form a structure resembling a tRNA,that is, having structure as described below. The specific sequence ofthe hydrogen bonded regions is not as critical, as long as the desiredstructure is formed. All tRNAs, including tRNAs from a wide variety ofbacteria and eukaryotes, conform to the same general secondarystructure. This is typically written in the form of a cloverleaf,maintained by hydrogen-bonded base pairing between short complementaryregions. The four major arms are named for their 10 structure orfunction: The acceptor arm consists of a 3' terminal CCA_(OH) plus avariable fourth nucleotide extending beyond the stem formed bybase-pairing the 5' and 3' segments of the molecule. The other armsconsist of base-paired stems and unpaired loops. The "T" arm is namedfor the presence of the ribothymidine nucleotide and contains sevenunpaired bases in the loop. The anticodon arm always contains theanticodon triplet in the center of the loop and consists of sevenunpaired bases. The D arm is named for the presence of the basedihydrouridine in the loop, another of the chemically modified bases intRNA, and includes between eight and twelve unpaired bases. Positionsare numbered from 5' to 3' according to the most common tRNA structure,which has 76 residues. The overall range of tRNA lengths is from 74 to95 bases. The variation in length is caused by differences in thestructure of two of the arms, the D arm and the extra or variable arm,which lies between the T and anticodon arms, which can contain betweenthree and five bases, or between 13 and 21 bases with a stem of aboutfive bases. The base pairing that maintains the secondary structure isvirtually invariant: there are always seven base pairs in the acceptorstem, five in the T arm, five in the anticodon arm, and three or four inthe D arm.

As used herein, a hybrid structure, consisting of an EGS hydrogen bondedto an RNA substrate, having secondary structure resembling a precursortRNA under conditions promoting cleavage by RNase P of the substrate atthe nucleotide at the 5' end of the base-paired region, preferablyincludes a D stem, an aminoacyl stem, and a T loop and stem, where thesequence of the latter may be changed compared to the sequence anddetailed structure found in the parent molecule.

A few nucleotides are always found in the same positions in 90 to 95% oftRNAs, with some additional nucleotides being semiconserved orsemivariant. This is not an absolute requirement in the GS, as long asthe sequence is complementary to the target and forms the secondarystructure characteristic of the tRNA. In fact, the sequence forming theaminoacyl stem and D loop and stem are changed in the GS to becomplementary to the target RNA.

The base paired double-helical stems of the secondary structure aremaintained in the tertiary structure, creating two double helices atright angles to each other. The acceptor stem and the T stem form onecontinuous double helix with a single gap; the D stem and the anticodonstem form another continuous double helix, also with a gap. Many of theinvariant and semi-invariant bases are involved in the tertiarystructure.

The complementary sequences will generally consist of elevennucleotides, or, under certain conditions may consist of as few as sevennucleotides, in two blocks which base pair with the target sequence andwhich are separated by two unpaired nucleotides in the target sequence,preferably UU, wherein the two blocks are complementary to a sequence 3'to the site targeted for cleavage.

B. Prokaryotic RNase P Targeting Sequence

The requirements for a GS functional with prokaryotic RNase P are lessstringent than those for a eukaryotic GS. The critical elements of aprokaryotic GS are (1) nucleotide sequence which specifically binds tothe targeted RNA substrate to produce a short sequence of base pairs 3'to the cleavage site on the substrate RNA and (2) a terminal 3'-NCCA,where N is any nucleotide, preferably a purine. The sequence generallyhas no fewer than four, and more usually six to fifteen, nucleotidescomplementary to the targeted RNA. It is not critical that allnucleotides be complementary, although the efficiency of the reactionwill vary with the degree of complementarity. The rate of cleavage isdependent on the RNase P, the secondary structure of the hybridsubstrate, which includes the targeted RNA and the presence of the3'-NCCA in the hybrid substrate.

Ribonuclease P

Ribonuclease P is an enzyme consisting of protein and RNA subunits thatcleaves tRNA precursors to generate the 5' termini of tRNAs. Thisessential enzymatic activity has been found in all cell types examined,both prokaryotic and eukaryotic. During the studies on recognition ofsubstrate by RNase P, it was found that E. coli RNase P can cleavesynthetic tRNA-related substrates that lack certain domains,specifically, the D, T and anticodon stems and loops, of the normal tRNAstructure. A half-turn of an RNA helix and a 3' proximal CCA sequencecontain sufficient recognition elements to allow the reaction toproceed. The 5' proximal sequence of the RNA helix does not have to becovalently linked to 3' proximal sequence of the helix. The 3' proximalsequence of the stem can be regarded as a "guide sequence" because itidentifies the site of cleavage in the 5' proximal region through abase-paired region.

RNase P from E. coli and human cells have similar but not identicalbiochemical properties. Their RNA components have similar secondarystructures. However, the substrate range of human RNase P is muchnarrower than that of the E. coli enzyme. For example, although E. coliRNase P can cleave a synthetic tRNA-related substrate that lacks threespecific domains of the normal tRNA structure, the human enzyme and thestructurally similar enzyme from the yeast, S. cerevisiae, cannot cleavethe same substrate. However, the E. coli RNase P can cleave a synthetictRNA-related substrate that is also cleaved by the human RNase P. Altmanet al., Genomics 18: 419422 (1993), describes several mammalian RNase Pcatalytic RNAs and identifies common features and differences.

As used herein, unless otherwise specified, RNase P refers to the RNaseP in the cell in which the RNA to be cleaved is located, whetherendogenous, added to the cell, or as used in vitro. Many of thetechniques described herein are known to those skilled in the art, asare methods for making, and sources of, reagents. The teachings of anyreferences cited herein with respect to methods and reagents arespecifically incorporated herein, as well as for the purpose ofdemonstrating the scope and level of skill in the art.

It is not necessary to provide RNase P activity if the cleavage is tooccur in bacterial cells or intracellularly in the nucleus since alleukaryotic cells contain RNase P in their nuclei. RNase P must besupplied if cleavage is to occur in the cytoplasm of eukaryotic cells.As used herein for ease of convenience, RNase P refers to theribonucleoprotein consisting of prokaryotic or eukaryotic analogues ofthe E. coli CS protein and M1 RNA, regardless of source, whetherisolated, or produced by chemical synthesis. The RNA subunit of RNase Palso can be transcribed from a gene. The eukaryotic RNase P RNA subunitis referred to as H1 RNA. The RNA subunit need not necessarily manifestcatalytic activity in the absence of protein subunits in vitro.

A. Endogenous RNase P

The sequence and proposed secondary structure of H1 RNA, the RNAcomponent of human RNase P, was reported by Altman et al. 25 (1993), theteachings of which are generally known in the art. The sequence andproposed structure of H1 RNA is shown in FIG. 8 (Sequence ID No. 11).The sequence and proposed secondary structure of M1 RNA, the RNAcomponent of E. coli RNase P, was reported by James et al., Cell 52: 19(1988), the teachings of which are generally 30 known in the art. Thesequence of M1 RNA is included as Sequence ID No. 40.

Because of the similarity in secondary structure and substratespecificity among the RNase P's of diverse origin, it is possible to usean EGS designed to maximize efficiency of cleavage for the RNase P inquestion using techniques described herein to target any RNA in anycell, even though the catalytically active RNA subunits may havedistinctly different sequences. See Altman, Ann. Rev. Enzymology 62:1-39 (1989); Altman, J. Biol. Chem. 265: 20053-20056 (1990). Secondarystructure is defined by intramolecular associations of complementarysequences. Base pairs can be canonical, A/U and G/C, or non-canonical,G/U, A/G, etc.

B. Exogenous RNA Having Catalytic Activity

An EGS can also be used in combination with an RNA sequence thatdemonstrates enzymatic activity in the presence or absence of a protein.That RNA sequence can be represented by a molecule like the entire H1 orM1 RNA molecule, any functionally equivalent molecule of prokaryotic oreukaryotic origin or derivation, or any portion thereof shown to havecatalytic activity, either alone or in combination with a protein. Sucha catalytic RNA is referred to herein as RNase P catalytic RNA and itssequence is referred to as an RNase P catalytic sequence. An RNA asdescribed above is considered an RNase P catalytic RNA regardless ofsource, whether isolated, produced by chemical synthesis, or transcribedfrom a gene. As noted above, an EGS effective to convert a targetedsequence into a substrate for human RNase P, will also be effective inmaking the substrate a target for procaryotic RNase P.

RNase P catalytic RNA can be derived from naturally occurring RNase Pcatalytic RNAs, for example, by deleting portions and by makingnucleotide substitutions. Such derived catalytic RNAs need only retainenough of the catalytic activity of naturally occurring RNase Pcatalytic RNA to cleave target RNA. A preferred method of generatingRNase P catalytic sequences is by in vitro evolution as described below.

There are two principle situations in which RNase P catalytic RNA orRNase P is utilized in combination with EGS: in vitro in the absence ofcells or cellular RNase P and in circumstances wherein the RNA to becleaved is located in a portion of a cell not containing endogenousRNase P. In the latter case, the genes encoding the analogs of M1 RNAand C5 protein, as defined above, or the human or other eukaryoticequivalents thereof, are introduced into the cell at the desiredlocation for cleavage using a suitable vector or other method known tothose skilled in the art for introduction and expression of a gene in acell.

RNase P Internal Guide Sequences

A guide sequence and the catalytic RNA subunit of an RNase P can belinked to form a single oligonucleotide molecule possessing both thetargeting function of an EGS and cleavage function of RNase P catalyticRNA. Such a combination, in a single oligonucleotide molecule, isreferred to as an RNase P internal guide sequence (RIGS). An RIGS can beused to cleave a target RNA molecule in the same manner as EGS.

RIGSs can be formed by linking a guide sequence to an RNase P catalyticsequence by any suitable means. For example, an EGS and RNase Pcatalytic RNA can be prepared as separate molecules which are thencovalently linked in vitro. Alternatively, a complete RIGS can besynthesized as a single molecule, either by chemical synthesis, or by invitro or in vivo transcription of a DNA molecule encoding linked GS andRNase P catalytic sequence. The linkage between the GS and RNase Pdomains of an RIGS can have any form that allows the domains to cleave atarget RNA. For example, the two domains could be joined by anoligonucleotide linker. Preferably, the linker will be composed of anordinary nucleotides joined by phosphodiester bonds. The GS and RNase Pcatalytic sequence components can be joined in either order, with theRNase P catalytic sequence linked to either the 3' end or 5' end of theGS component.

RIGSs can be used for cleavage of target RNA both in vitro and in vivo.In vitro, the RIGS can function without RNase P protein components invitro, although activity of the RIGS can be increased by the addition ofRNase P protein components. In vivo, endogenous RNase proteins willstimulate activity of the RIGS. The activity of both prokaryotic- andeukaryotic-based RIGSs are expected to be enhanced by the presence ofeither prokaryotic or eukaryotic RNase P protein components.

Method For Producing EGSs And RIGSs Having Enhanced Efficacy

EGSs and RIGSs having enhanced binding affinity as measured by decreasedenergy of binding can be designed by in vitro evolution. Such a methodcan be used to identify RNA molecules with desired properties from poolsof molecules that contain randomized sequences. As demonstrated moreclearly in the examples, appropriately modified, these methods can beused for the isolation of efficient EGSs and RIGSs. These new EGSs, whencomplexed with an exemplary target RNA, CAT mRNA substrate (Sequence IDNo. 7), allow cleavage of the target by human RNase P at rates similarto those achieved with natural substrate.

The general selection scheme is depicted in FIG. 2. In each round ofselection, the pool of RNAs is digested with human RNase P, or with theRIGS, and the cleaved products are isolated by electrophoresis and thenamplified to produce progeny RNAs. One of the template-creatingoligonucleotides is used as the 5' primer for the polymerase chainreaction (PCR) in order to allow restoration of the promoter sequenceand the leader sequence of the chimeric RNA for the next cycle ofselection. The stringency of selection is increased at each cycle byreducing the amount of enzyme and the time allowed for the cleavagereaction, such that only those substrates that are cleaved rapidly bythe enzyme are selected.

In the first three rounds of selection, RNA substrates are digested withan appropriate amount of human RNase P, for example, 3.6 units, or theequivalent activity of the RIGS. One unit of human RNase P is defined asthat amount of enzyme that cleaves 1 pmol of precursor to tRNA_(Tyr)from E. coli in 30 min at 37° C. For assays in subsequent rounds ofselection, the amount of enzyme is reduced, and the incubation time isshortened so that less than 20 percent of the substrate is cleaved.Cleavage products are separated from uncleaved substrates byelectrophoresis and RNA extracted.

The purified cleavage product RNAs are reverse transcribed and amplifiedby PCR. The double-stranded DNA generated by PCR regains the promotersequence and the leader sequence from the sequence in the primer, and isthen used as a template for transcription of RNA for the next round ofselection. After eight cycles of selection, the resulting pool ofdouble-stranded DNAs is cloned into an appropriate vector and sequenced.

In order to test the abilities of EGSs or RIGSs derived from theindividual variants, sequences corresponding to the GS segment of eachchimeric tRNA are amplified by PCR, and RNAs transcribed with anappropriate RNA polymerase. RNA cleavage by the selected EGS or RIGS isthen assayed. Sequences in common in the most active EGS and RIGSs arethen determined and new EGS and RIGSs designed.

As described in the examples, simulation of evolution in vitro was usedto select EGSs that bind strongly to a target substrate mRNA and thatincrease the efficiency of cleavage of the target by human ribonucleaseP to a level equal to that achieved with natural substrates. The mostefficient EGSs from tRNA precursor-like structures with the target RNA,in which the analog of the anticodon stem has been disrupted, anindication that selection for the optimal substrate for ribonuclease Pyields an RNA structure different from that of present-day tRNAprecursors.

Chemically Modified EGS And RIGS

Although chemically unmodified oligoribonucleotides can function aseffective EGS or RIGS in a nuclease-free environment, the shorterhalf-life in serum and inside cells reduces their effectiveness astherapeutics. Chemical modifications can be made which greatly enhancethe nuclease resistance of EGS and RIGS without compromising theirbiological function of inducing or catalyzing cleavage of RNA target.For example, one or more of the bases of an EGS or RIGS construct can bereplaced by 2' methoxy ribonucleotides or phosphorothioatedeoxyribonucleotides using available nucleic acid synthesis methods.Synthesis methods are described by, for example, Offensperger et. al.,EMBO J. 12: 1257-1262 (1993); PCT WO 93/01286 by Rosenberg et al.(synthesis of sulfurthioate oligonucleotides); Agrawal et al., Proc.Natl. Acad. Sci. USA 85: 7079-7083 (1988); Sarin et al., Proc. Natl.Acad. Sci. USA 85: 7448-7794 (1989); Shaw et al., Nucleic Acids Res 19:747-750 (1991) (synthesis of 3' exonuclease-resistant oligonucleotidescontaining 3' terminal phosphoroamidate modifications); all of which arehereby incorporated herein by reference.

It is well documented in the current literature that degradation ofoligonucleotide analogues is mainly attributable to 3'-exonucleases.Several studies have also demonstrated that various 3'-modifications cangreatly decrease the nuclease susceptibility of these analogues. Thus,another method to reduce susceptibility to 3' exonucleases isintroduction of a free amine to a 3' terminal hydroxyl group of the EGSor RIGS molecule, as described, for example, by Orson et al., Nucl.Acids Res. 19: 3435-3441 (1991). Furthermore, cytosines that may bepresent in the sequence can be methylated, or an intercalating agent,such as an acridine derivative, can be covalently attached to a 5'terminal phosphate to reduce the susceptibility of a nucleic acidmolecule to intracellular nucleases. Examples of this are described inMaher et al., Science 245: 725-730 (1989) and Grigoriev et al., J. Biol.Chem. 267: 3389-3395 (1992).

Another class of chemical modifications is modification of the 2' OHgroup of a nucleotide's ribose moiety, which has been shown to becritical for the activity of the various intracellular and extracellularnucleases. Typical 2' modifications are the synthesis of 2'--O--Methyloligonucleotides, as described by Paolella et al., EMBO J. 11: 1913-1919(1992), and 2'-fluoro and 2'-amino-oligonucleotides, as described byPieken et al., Science 253: 314-317 (1991), and Heidenreich andEckstain, J. Biol. Chem 267: 1904-1909 (1992). Portions of EGS and RIGSmolecules can also contain deoxyribonucleotides. Such substitutionsimprove nuclease resistance by eliminating the critical 2' OH group.

Application Of EGS And RIGSs As Laboratory Or Clinical Reagents

External guide sequences and RIGSs have applications as in vitroreagents, in a similar fashion to restriction enzymes, and astherapeutic agents, for cleavage and inactivation of specific host cellRNA or RNA coded for by pathogenic organisms such as bacteria orviruses, as demonstrated by the following examples.

As used herein, an EGS or RIGS is an oligonucleotide molecule. It is tobe understood, however, that for therapeutic purposes, a DNA moleculeencoding an EGS molecule or encoding an RIGS molecule could be utilized.Accordingly, unless otherwise specified, administration of an EGS orRIGS encompasses both the RNA molecule that hydrogen bonds to a targetnucleic acid sequence that is cleaved, as well as a DNA moleculeencoding the RNA molecule, which is expressed under conditions whereinthe RNA molecule functions as an EGS or RIGS.

An external guide sequence can be brought into contact with any RNAhaving sequences complementary to the EGS, in the presence of RNase P,and the RNA will be cleaved at the targeted site. In this manner, theactivity of endogenous RNase P in any cell, such as the RNase P of humancells, can be directed to destroy specific messenger, viral or otherRNAs by the use of an appropriate EGS RNA.

A. Reagents For In vitro Applications

DNA restriction endonucleases are invaluable reagents for the molecularbiologist. Patterns of restriction fragment sizes are used to establishsequence relationships between DNA molecules, and large DNAs can becleaved to give fragments of sizes useful for genetic engineering,sequencing, and studying protein binding. RNA processing enzymes can beutilized under conditions such that they also cleave RNA withconsiderable sequence specificity.

Specific ribozymes can be prepared by combining the specific guidesequence with RNase P or functional equivalents thereof. In thepreferred embodiment, the external guide sequence and the RNase Pcatalytic RNA are separate; alternatively, the two sequences can bejoined either directly or via a linker. The linker can be any moleculethat can be covalently bound to oligonucleotides. Numerous linkers areknown to those of skill in the art. A preferred linker is anoligonucleotide because it allows direct synthesis of the complete RIGS.

B. Therapeutics

1. Determination and Preparation of Complementary Sequences

Any cellular gene product expressed as RNA, including proteins encodedby mRNA and structural RNAs themselves, can be targeted for inactivationby RNase P, or directly by an RIGS using sequences engineered to includeappropriate regions of sequence and/or structure for binding to thetargeted RNA and the desired site of cleavage. The cellular gene productcould be a product of an oncogene with an altered sequence, such as theras gene product; where the product is not a normal cell component, aviral protein, such as one encoded by an essential gene for HIVreplication; or a bacterial protein.

In many cases, the critical genes an infective or pathological agenthave been isolated and sequenced. Appropriate complementary sequencescan be synthesized using standard techniques, reagents, and equipmentbased on these known sequences.

2. Preparation of an appropriate pharmaceutical composition for deliveryof the EGS or RIGS to the targeted RNA.

There are two primary mechanisms for delivering the EGS or RIGS tointracellular RNA that has been targeted for cleavage: diffusion and viaa vector.

As discussed above, any RNA that is important in a disease process canbe targeted and appropriate complementary sequences made syntheticallyor by copying cloned sequence. For example, cancer regulatory genes canbe targeted. Since RNase P is predominantly found in the nucleus ofeukaryotic cells, the infectious agents most likely to be inhibited byadministration of appropriate EGS to the infected cells are those inwhich critical RNA sequences are transcribed in the nucleus. Importantexamples of the viral agents that replicate in the cell nucleus includeherpes viruses, including herpes simplex virus, varicella-herpes zostervirus, cytomegalovirus, and Epstein-Barr virus; hepatitis B virus;adenoviruses; paramyxoviruses such as measles; and the retroviruses,such as human immunodeficiency virus, HIV I, HIV II, HIV III and HTLV-1.RIGSs should cleave target RNA in any area of the cell since thecatalytic activity is self-contained.

3. Vector-mediated delivery of EGS and RIGSs.

Preferred vectors are viral vectors such as the retroviruses whichintroduce EGS and RIGS directly into the nucleus where it is transcribedand released into the nucleus. Under the appropriate conditions, the EGSor RIGS will hybridize to the targeted RNA and the endogenous RNase P orRIGS will cleave the hybridized RNA at the 5' side of the hybrid region.

Methods for using retroviral vectors for gene therapy are described inU.S. Pat. Nos. 4,868,116 and 4,980,286, and PCT applicationPCT/US89/03794 and PCT/US89/00422, the teachings of which areincorporated herein.

Defective retroviral vectors, which incorporate their own RNA sequencein the form of DNA into the host chromosome, can be engineered toincorporate EGS and RIGSs into the host, where copies will be made andreleased into the cytoplasm to interact with the target nucleotidesequences.

EGS and RIGSs are particularly useful as a means of targeted therapyinto hematopoietic cells of patients suffering from virus-induceddisease of those cells, such as AIDS. The most efficacious methodologypresently available for the introduction of specific genetic sequencesinto human cells involves the use of RNA-containing retroviruses whichserve as vehicles or vectors for high efficiency gene transfer intohuman cells.

RNase P-based therapy can also be used as a means of preventing thespread of HIV-1 and or providing a HIV-1 resistant population of T-cellsthat will be able to confer immune function to infected individuals.Patients who have been recently diagnosed as having antibodies to HIV-1,but who do not yet show AIDS symptomatology, are the preferredcandidates for therapy. This procedure necessitates removal of some ofthe patient's bone marrow stem cells and subsequent partial cytoblation.The removed cells can be treated in the laboratory with appropriate EGSor RIGS compositions, using appropriate viral vectors, such as defectiveviral vectors, and then restored to the same individual. The treatedcells will develop in the patient into mature hematopoietic cells,including T-cells. These T-cells will have normal immune function and,most importantly, will be intracellularly immunized to prevent theirdestruction by any HIV-1 still present in the patient.

Bone marrow stem cells and hematopoietic cells are relatively easilyremoved and replaced and provide a self-regenerating population of cellsfor the propagation of transferred genes. RNase P-based therapeuticsallow the selective inactivation of other unwanted genes in cells, suchas activated oncogenes, involved in the causation and maintenance ofcancer cells.

In contrast to the approaches presently in use which are aimed atpreventing or limiting infection with HIV, it should be possible to useRNase P-based technology to treat, and possibly to cure, HIV infection,and related diseases of white blood cells which are subject totransformation by retroviral vectors carrying EGS or RIGS. Particularexamples of diseases that may be treated using EGS and RIGS include notonly HTLV-1, but also various retroviral-induced leukemias resultingfrom chromosomal translocations that produce chimeric RNAs which produceproteins that are unique to those cells and that can act as growthstimulators or oncogenes. Other types of transformed tissues that mightbe treatable include all cancer cells carrying identified oncogenes ofknown sequence.

4. Topical and other EGS and RIGS compositions for local administration.

EGS and RIGS may also be administered topically, locally or systemicallyin a suitable pharmaceutical carrier. Remington's PharmaceuticalSciences, 15th Edition by E. W. Martin (Mark Publishing Company, 1975),the teachings of which are incorporated herein by reference, disclosestypical carriers and methods of preparation. EGS and RIGS may also beencapsulated in suitable biocompatible microcapsules, microparticles ormicrospheres formed of biodegradable or non-biodegradable polymers orproteins or liposomes for targeting to phagocytic cells. Such systemsare well known to those skilled in the art and may be optimized for usewith the appropriate EGS and RIGSs.

Therapeutically the oligoribonucleotides are administered as apharmaceutical composition consisting of an effective amount of the EGSor RIGS to inhibit transcription of a targeted RNA and apharmaceutically acceptable carrier. Examples of typical pharmaceuticalcarriers, used alone or in combination, include one or more solid,semi-solid, or liquid diluents, fillers and formulation adjuvants whichare non-toxic, inert and pharmaceutically acceptable. Suchpharmaceutical compositions are preferable in dosage unit form, that is,physically discreet units containing a predetermined amount of the drugcorresponding to a fraction or multiple of the dose which is calculatedto produce the desired therapeutic response. It is essential that theoligonucleotides be delivered in a form which prevents degradation ofall of the oligonucleotide before it reaches the intended target site.

A preferred embodiment is an EGS or an RIGS administered as a viralvector, encoding the EGS or RIGS, or in a liposome, such that aneffective amount of EGS or RIGS is delivered. Generally these willproduce a concentration between 1 μM and 1 mM at the site of the cellsto be treated. Such compounds and compositions can be formulated as atopical composition, for example, for application to a viral lesion suchas that produced by herpes simplex virus. These will generally containbetween 1 μM and 1 mM oligonucleotide per unit of carrier, or produce aconcentration between 1 μM and 1 mM at the site of the cells to betreated. Oral compositions, although not preferred, are in the form oftablets or capsules and may contain conventional excipients. Anotherpreferred composition is a polymeric material applied locally forrelease of EGS or RIGS. Still another preferred composition is asolution or suspension of the EGS or RIGS in an appropriate vector incombination with conventional pharmaceutical vehicles are employed forparenteral compositions, such as an aqueous solution for intravenousinjection or an oily suspension for intramuscular injection.

For clinical applications, the dosage and the dosage regimen in eachcase should be carefully adjusted, utilizing sound professional judgmentand consideration of the age, weight and condition of the recipient, theroute of administration and the nature and gravity of the illness.

The present invention, EGS and RIGSs, will be further understood byreference to the following non-limiting examples.

EXAMPLE 1 Cleavage of tRNA precursor fragments by human RNase P in thepresence of EGS.

An EGS that can target RNA for cleavage by human RNase P was preparedusing a small RNA fragment, pAva (Sequence ID No. 6, FIG. 3) whichcontains a 5' precursor sequence and the first fourteen nucleotides fromthe 5' terminus of a tRNA. The leader sequence of E. coli tRNA^(Tyr)precursor can be cleaved correctly when another piece of RNA, forexample, EGSΔ1-18 (Sequence ID No. 5), which lacks the first eighteennucleotides of the 5' terminus of mature tRNA^(Tyr) but retains theremaining 3' proximal sequence, is hybridized to the target RNA.

Human RNase P was partially purified from HeLa cells using the method ofBartkiewicz et al., Genes and Development 3: 488-499 (1989). Thesubstrates were prepared by in vitro transcription in the presence ofα-³² P!GTP. α-³² P!GTP labelled pAva (Sequence ID No. 6) I RNA (28 nt)was mixed with unlabelled EGS RNA and the mixture was incubated at 37°C. in 50 mM Tris-Cl (pH 7.5), 100 mM NH₄ Cl, and 10 mM MgCl₂ with enzymefor 30 min. Labelled pAva (Sequence ID No. 6) I RNA alone was alsoincubated with or without enzyme. Analysis by gel electrophoresis showsthat the EGS plus enzyme resulted in cleavage of the RNA.

The 3' proximal oligonucleotide is the external guide sequence. Becausethe lengths of the leaders and their sequences, as well as the sequencesof the mature domain, are not conserved among different precursor-tRNAs,the main determinants for human RNase P cleavage must be in some of theconserved structural features of various tRNAs. This general idea isborne out by the fact that several other EGSs that did not mimic exactlythe structure of parts of a tRNA did not target complementary RNAs.Examples of such changes include changes in the number of possible basepairs in the D or amino acyl stems, changes in positions 8 and 9 of themature tRNA sequence, and a change from cytosine to uracil at position57. However, an EGS that lacked the anticodon stem and loop, or thevariable stem and loop, resulted in efficient cleavage, indicating thatthese parts of the EGS, separately, were not essential for recognitionof the target complex by the enzyme.

Accordingly, if an mRNA, rather than part of a precursor tRNA sequence,is incorporated into the double-stranded stem region of a putativetarget complex, and the resulting hybrid contains the structuralfeatures required of a substrate for human RNase P activity, the mRNAwill be cleaved by human RNase P.

EXAMPLE 2 Specific Cleavage of CAT mRNA in vitro by Human RNase P usingan External Guide Sequence

Examples provided below show the efficiency of cleavage of the mRNA forchloramphenicol acetyltransferase (CAT) by human RNase P. The sequenceof the 5' oligoribonucleotide, as well as that of the EGS, depends onthe choice of target site in the mRNA. The presence of the appropriatelydesigned EGS efficiently reduces CAT enzymatic activity in vivo, as wellas promotes cleavage of CAT mRNA in vitro, indicating that this methodshould be of general use for gene inactivation.

An EGS custom-designed for the mRNA for chloramphenicol transacetylase(CAT), shown in FIG. 4A, EGS^(CAT), Sequence ID No. 8), can direct thespecific cleavage of CAT mRNA by human RNase P in vitro or in vivo cellsin tissue culture. However, the cleavage reaction is inefficientcompared to cleavage of natural tRNA precursor substrates.

The proposed secondary structure of a complex of CAT mRNA (Sequence IDNo. 7) and EGS^(CAT) (Sequence ID No. 8) resembles the tRNA cloverleafstructure, but it includes sequences not normally found in the tRNA fromwhich it was originally derived, namely, tyrosyl tRNA (tRNA^(Tyr))(Sequence ID No. 6) of Escherichia coli. To ensure that appropriatetertiary interactions that facilitate the process of enzyme-substraterecognition exist in the complex, parts of the EGS that participate intertiary interactions in the analogous tRNA structures were changed intwo ways. First, four nucleotides in the equivalent of the T loop andfive in the equivalent of the variable loop were randomized byincorporation of equimolar quantities of the deoxyribonucleotides dA,dG, dC and T into a DNA template to yield an initial population of2.6×10⁵ sequence variants. Second, during each round of selectiveamplification, random mutations were introduced by performing polymerasechain reaction (PCR) at an error rate of approximately 0.1 percent pernucleotide incorporated, using the method of A. Beaudry and G. F. Joyce,Science 257: 635 (1992). The mRNA for the gene for chloramphenicolacetyltransferase (CAT) can be easily manipulated on plasmids and theenzymatic activity is readily expressed in tissue culture cells so itwas used as a target substrate. As demonstrated below, an EGS can targetCAT mRNA (Sequence ID No. 7) for specific cleavage by human RNase P.FIG. 4A shows a complex in which an EGS, EGS^(CAT) (Sequence ID No. 8),could base-pair with nucleotides 67 to 79 of CAT mRNA (Sequence ID No.7), where the first nucleotide of the translation initiation codon isnumbered 1, and direct human RNase P to cleave that mRNA at nucleotide67. The EGS^(CAT) (Sequence ID No. 8) construct was derived from the E.coli tRNA^(Tyr) (Sequence ID No. 6) gene where the first eighteennucleotides from the 5' terminus have been deleted and the sequences onthe D-loop and acceptor-loop have been changed to make base-pairs withCAT mRNA. The EGS^(CAT) (Sequence ID No. 8) fused upstream with a T7promoter was cloned into a pUC19 vector. The EGS^(CAT) RNA (Sequence IDNo. 8) was prepared through in vitro transcription with T7 RNApolymerase.

The HindIII-BamHI fragment of CAT gene (pCAT™, Promega) was cloned inpGem-2. The plasmid was truncated with EcoRI and a 260 nucleotide-longtranscript was obtained by in vitro transcription with T7 RNA polymerasein the presence of α-³² P!GTP. The EGS sequence was synthesized bypolymerase chain reaction, using the E. coli tRNA^(Tyr) gene astemplate, with oligonucleotide GCCAAACTGAGCAG ACTC (Sequence ID No. 12)and GCGCggtaccAAAAATGGTGAGG CATGAAGG (Sequence ID No. 13). The boldletters in the oligonucleotide sequences indicate the bases needed tomake base pairs to CAT mRNA, the underlined letters indicate thesequence complementary to the transcription termination signal, and thelower case letters shows an extra linker sequence. The sequence GCGC atthe 5' end of the second oligonucleotide are extra nucleotides. The PCRfragment was digested with HindIII and cloned into pUC19 with a T7promoter upstream from the EGS sequence. The EGS^(CAT) RNA (Sequence IDNo. 8) was transcribed with T7 RNA polymerase after the plasmid waslinearized with DraI. A mixture of unlabelled and α-³² P!GTP labelledCAT mRNA fragment, 0.2 pmole in total, was mixed with the EGS^(CAT) RNA(Sequence ID No. 8) in amounts of 4 pmole, 1 pmole, 1 pmole, 0.4 pmoleand 0.2 pmole. Each mixture was incubated at 37° C. in 50 mM Tris-Cl (pH7.5), 100 mM NH₄ Cl, and 25 mM MgCl₂ with enzyme for one hour. Thereaction was stopped by addition of an equal volume of dye solution withexcess EDTA and then subjected to a 5 % polyacrylamide-7M urea gel. CATmRNA alone was incubated without and with enzyme and loaded on the gel.

Primer extension analysis determining the precise site of EGS^(CAT)-directed cleavage by human RNase P was conducted as follows. A reversetranscription reaction was performed on the uncleaved and cleaved CATmRNA using an oligodeoxyribonucleotide GGCCGTA ATATCCAGCTGAACGG(Sequence ID No. 14), complementary to nucleotides 129 to 107 of CATmRNA. The reaction was incubated in 100 mM Tris-Cl (pH 8.3), 10 mM KCl,6 mM MgCl₂, 10 mM DTT and 2 units AMV reverse transcriptase at 46° C.for 2 hours. Labelled G, A, U, C were used as reference analyses of DNAsequences corresponding to the CAT mRNA template.

The precise site of cleavage of CAT mRNA was determined by primerextension analysis using an oligodeoxyribonucleotide primercomplementary to nucleotides 129 to 107 of the RNA, showing that thecleavage occurs between nucleotides 66 and 67, as expected.

The results of the EGS^(CAT) RNA-directed cleavage of CAT mRNA wereanalyzed by gel electrophoresis. In the presence of EGS^(CAT) (SequenceID No. 8) molecules, CAT mRNAs were cleaved to give rise to two productswith the expected size. Analysis of the products of the reaction showedthat the end groups contained 5' phosphoryl and 3' hydroxyl termini, thesame as those normally generated by RNase P. The results showconclusively that the specific cleavage of CAT mRNA is due to anEGS-directed RNase P hydrolytic reaction.

For up to five-fold molar excess of EGS^(CAT) RNA to mRNA, the cleavageefficiency is proportional to the amount of EGS^(CAT) added. However,more than ten-fold excess of EGS^(CAT) molecules caused a decrease inthe cleavage efficiency. One explanation for this is that EGS^(CAT)alone inhibits the enzymatic activity by competing with the mRNA-EGScomplex for the enzyme. The reaction proceeds in a linear fashion formore than 3 hours at 37° C. Denaturation and reannealing of theoligonucleotides in the target complex did not improve the efficiency ofcleavage. The reaction has an absolute requirement for Mg²⁺ with anoptimal concentration of 25 mM, in contrast to that with tRNA^(Tyr)precursor as substrate, which has an optimal Mg²⁺ concentration ofbetween 2 and 10 mM.

EXAMPLE 3 Inhibition of Expression of CAT Activity in Green Monkey CV-1Cells by EGS^(CAT)

In order to test whether the EGS can function in vivo, the EGS^(CAT)sequence (Sequence ID No. 8) was inserted downstream of a mouse U6 snRNAgene promoter in a BLUESCRIPT™ (Stratagene, La Jolla, Calif.) vectorforming pEGS^(CAT). The EGS^(CAT) sequence (Sequence ID No. 8) can betranscribed by RNA polymerase III and the transcription can terminate ata T₅ cluster following the EGS sequence in either S100 extract or livingcells. Green monkey fibroblast cells CV-1 were cotransfected with pCATand pEGS^(CAT) plasmids. After transfection with pCAT, which encodes theCAT gene, and PEGS^(CAT), cells were harvested and CAT activity wasassayed.

CV-1 cells were maintained in Dulbecco's modified Eagle medium thatcontained 10% fetal calf serum. One day prior to transfection cells weresplit 1:10 and plated in 60 mm Petri plates. Two hours prior totransfection cells were fed with 4.5 ml of fresh medium with 10% fetalcalf serum. Transfection was performed by the calcium phosphateprecipitation procedure using 2.5 μg of pCAT DNA and various amounts ofPEGS^(CAT) DNA, ranging from one to 6.25 μg. Twenty-four hours aftertransfection, cells were harvested and cell extracts were assayed forCAT activity.

The extract from cells cotransfected with EGS^(CAT) construct apparentlydecreased the conversion of chloramphenicol to its acetylated forms. Thedegree of inhibition was measured quantitatively by counting of thespots excised from a TLC plate. EGS^(CAT) cotransfection producedgreater than 50% inhibition compared to the control with no EGS^(CAT)cotransfection. There was substantial loss of ability to inhibit CATexpression when a higher ratio of pEGS^(CAT) to pCAT was introduced.Similar experiments yielding approximately 70% of CAT activity have alsobeen performed with human cells in tissue culture.

EXAMPLE 4 Preparation of EGS with Altered Sequence that EnhanceDegradation of Target RNA by RNase P

As explained in detail below, two classes of EGS were designed, as shownin FIGS. 4A, 4B, 4C, 4D, 4E, and 4F. The first class involves deletionsof large segments of the EGS as suggested in Example 1 and described inExamples 2 and 3. For example, it has been found that the anticodon loopand stem can be deleted, as shown in EGS^(CAT) ΔAC (Sequence ID No. 10)of FIG. 4C and 4F, and the EGS can still promote cleavage by RNase P ofa target RNA (CAT mRNA containing Sequence ID No. 7 in FIGS. 4C and 4F).The anticodon loop and part of the variable loop can alternatively bedeleted from the EGS, and the EGS will promote cleavage with greaterefficiency than the parent EGS molecule. The most efficient EGS of thisdeletion class was the one in which the anticodon stem and loop wasdeleted. This EGS^(CAT) ΔAC (Sequence ID No. 10) promoted cleavage byhuman RNase P of a target mRNA (CAT mRNA) at a rate 10-fold higher thanthe parent EGS. The deletion of both variable and anticodon stems andloops, however, does not yield a more efficient EGS, although thevariable loop can consist of only one or two nucleotides and still behighly efficient.

The second class of EGSs have changes in both the equivalent of the Tloop, the variable loop, and the anticodon stem of the tRNA-like segmentof the EGS. Three such EGSs, described below, are EGS 6, EGS 8 and EGS 9(Sequence ID No. 9, FIGS. 4B and 4E). As shown below, EGS 9 is the mostefficient of the EGSs in these examples and directs cleavage by humanRNase P of a target RNA (CAT mRNA) at a rate approximately fifty to onehundred fold greater than the parent EGS.

These results apply to relative rates of cleavage at a particular sitein a target mRNA. The absolute rates of cleavage at any particular sitestill depend on access of the EGS to that particular site.

EXAMPLE 5 Preparation of RNAs with Randomized Nucleotides

A. Preparation of Chimeric Covalently Linked mRNA-EGS Substrate

The procedure to select for EGSs that are more efficient in guidingRNase P to the target CAT mRNA involves the synthesis of a population ofchimeric, covalently linked mRNA-EGS substrates which are then runthrough cycles of in vitro mutation and selection for molecules whichcan serve as substrates for RNase P. Double-stranded DNA templates weremade by annealing of and enzymatically extending two overlappingsynthetic oligonucleotides: TAATACGACTCACTATAGAACATTTTGAGGCATTTCAGTCAGTTGGCCAAACTGAGCAGAC (SEC-1A, Sequence ID No. 2)and TGGTGAGGCATGAAGGNN NNGAACCTTCNNNNNGCAGATTTAGAGTCTGCTCAGTTTGGCC(SEC-1B, Sequence ID No. 3), where the complementary sequences areunderlined and the randomized nucleotides (N) were introduced duringtheir machine synthesis by incorporating equimolar quantities of fournucleotides. These sequences create a chimeric tRNA gene which containssequences from CAT mRNA and tRNA^(Tyr) from E. coli as well as ninenucleotides (N) that are randomized. A promoter for T7 bacteriophage RNApolymerase is included in SEC-1A (Sequence ID No. 2). The extension wascarried out with AMV reverse transcriptase at 46° C. for two hours.Variant RNA pools were prepared by transcription with T7 polymerase in40 mM Tris Cl, pH 7.9, 6 mM MgCl₂ 10 mM dithiothreitol 2 mM spermidine 1mM NTPs containing 20 μci α-³² P!GTP at 37° C.

B. The Selection Procedure

The general selection scheme is described above. One of thetemplate-creating oligonucleotides (SEC-1A, Sequence ID No. 2) was alsoused as the 5' primer for the polymerase chain reaction (PCR) in orderto allow restoration of the T7 promoter sequence and the leader sequenceof the chimeric RNA for the next cycle of selection. The stringency ofselection was increased at each cycle by reducing the amount of enzymeand the time allowed for the cleavage reaction, such that only thosesubstrates that were cleaved rapidly by the enzyme were selected.

In the first three rounds of selection, RNA substrates were digestedwith 3.6 units of human RNase P, purified through the glycerol gradientstep described by Yuan and Altman, Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992) and Bartkiewicz et al., Genes and Dev. 3:488 (1989)) in50 mM Tris-Cl (pH 7.5), 10 mM MgCl₂, and 100 mM NH₄ Cl at 37° C. for 2hours. One unit of human RNase P is defined as that amount of enzymethat cleaves 1 pmol of precursor to tRNA^(Tyr) from E. coli in 30 min at37° C. For assays in subsequent rounds of selection, the amount ofenzyme was reduced, and the incubation time was shortened so that lessthan 20 percent of the substrate was cleaved.

Cleavage products were separated from uncleaved substrates byelectrophoresis on an eight percent polyacrylamide-7M urea gel. RNA wasextracted from the gels by the crush and soak method.

The purified cleavage product RNAs were reverse transcribed andamplified by PCR with SEC-1A (Sequence ID No. 2) and SEC-1C(TGGTGAGGCATGAAGG, Sequence ID No. 15) as primers with a Perkin ElmerRNA PCR kit. The double-stranded DNA generated by PCR regained the T7promoter sequence and the leader sequence from the sequence in theprimer SEC-1A, and it was then used as a template for transcription ofRNA for the next round of selection.

C. Characterization of the Selected RNAs and EGS RNA Derived from Them

After eight cycles of selection the resulting pool of double-strandedDNAs were cloned into the BLUESCRIPT™ vector (Stratagene, La Jolla,Calif.) vector. Eighteen plasmid DNAs were sequenced using Sequenase 2.0(U.S. Biochemicals, Cleveland, Ohio).

In order to test the abilities of EGSs derived from the individualvariants selected above for CAT mRNA targeting, sequences correspondingto the EGS segment of each chimeric tRNA were amplified by PCR usingprimers SEC-1A (Sequence ID No. 2) and SEC-1T(TAATACGACTCACTATAGGCCAACTGAGCAGAC, Sequence ID No. 16), which containsa promoter sequence for T7 polymerase, and RNAs were transcribed with T7RNA polymerase. EGS-directed CAT mRNA cleavage was assayed in 10 μl of50 mM Tris-Cl, pH 7.5, 10 mM MgCl₂ 100 nM NH₄ Cl containing 0.25 pmol(1000 cpm) of substrate RNA and 1 or 5 pmol of EGS RNAs. Reactionmixtures were incubated at 37° C. for 30 min with 10 units of RNase Pfrom HeLa cells, followed by electrophoresis in 5 % polyacrylamide/7Murea gels.

The gels showed a species of RNA migrating in the position expected forcleavage of substrate RNA in those lanes where the newly selected EGSshave been included in the reaction mixtures.

D. Sequence Analysis of Randomized EGSs.

Sixteen individual clones were sequenced. The sequence of the anticodonstem/loop, the variable (V) loop, and the T stem/loop are shown in FIG.6. From the sequence that had been randomized in the T-loop, twoparticular sequences were most frequently selected: UUCGUGC, found inseven clones, and UUCGCCC, also found in seven clones. The T loopsequences of the two remaining clones contained single transitionmutations of the two major sequences, UUCGUCC and UUCACCC. By contrast,no significant sequence-related bias was seen in the sequence of fivenucleotides in the variable loop.

In addition to the sequences in the T and variable loops that wereselected from the totally randomized sequence, a considerable number ofmutations were introduced into the EGS in the chimeric substrates as aconsequence of the conditions for PCR. Some of these mutations werebeneficial and, therefore, the sequences that included them wereselected and accumulated. In almost all of the individual selectedclones, the integrity of base-pairing in the anticodon stem wasdisrupted.

Table 1 shows the partial sequences of the anticodon loop/stem region,the variable (V) loop, and the T stem/loop region of the parent chimericmRNA-EGS^(CAT) substrate (P), nucleotides 28 through 88 of Sequence IDNo. 1. Table 1 also shows partial sequences of EGS segments of someindividual chimeric mRNA-EGS substrates, nucleotides 1 through 60 ofSequence ID No. 9 and Sequence ID Nos. 17 through 31, which wereobtained as a result of the in vitro selection procedure. Nucleotidesthat differ from the parent chimeric mRNA-EGS^(CAT) substrate sequenceare given in bold letters and are underlined. Hyphens indicatedeletions. The remaining part of the sequence of the mRNA-EGS chimericsubstrates is shown in FIG. 4A. Numbering of the partial sequences isnot uniformly consecutive, since some clones did not have appropriateinserts and only sixteen selected sequences are listed.

                                      TABLE 1    __________________________________________________________________________    Substrate #            D stem/   Anticodon           T stem/    (ID No.)            loop      stem/loop     V loop                                          loop    19      30        47            52    69            AA stem    __________________________________________________________________________    SCE-1   GGCCAAACUGA                      GCAGACUCUAAAUCUGC                                    NNNNN GAAGGUUCNNNNCCUUC                                                        AUGCCUCACCA    (28-88 of 1)    #1 (17) GGCCAAACUGA                      GCAGACUCUAAAUCGGC                                    CCUUC GAAGGUUCGCCCCCUUC                                                        AUGCCUCACCA    #2 (18) GGCCAAACUGA                      GCAGACUCUAAAUCUGC                                    ACGAGA                                          GAAGGUUCGUGCCCUUC                                                        AUGCCUCACCA    #4 (19) GGCCAAACUGA                      GCAGACUCUAAACUGGC                                    CUAAC GAAGGUUCGCCCCCUUC                                                        AUGCCUCACCA    #5 (20) GGCCAAACUGA                      GCAGACUCUAAAU-UGC                                    CCAAC GAAGGUUCACCCCCUUC                                                        AUGCCUCACCA    #6 (21) GGCCAAACUGA                      GCAGACUCCAAAUC--C                                    ACCAA GAAGGUUCGUGCCCUUC                                                        AUGCCUCACCA    #8 (22) GGCCAAACUGA                      GCAGACUCUAAA-CUCC                                    UCCCA GAAGGUUCGUGCCCUUC                                                        AUGCCUCACCA    #9      GGCCAAACUGA                      GCAGACUCUAAAUC-GC                                    AAACG GAAGGUUCGUGCCCUUC                                                        AUGCCUCACCA    (1-60 of 9)    #10 (23)            GGCCAAACUGA                      GCAGACUCUAAAUCGGC                                    CUACG GAAGGUUCGCCCCCUUC                                                        AUGCCUCACCA    #11 (24)            GGCCAAACUGA                      GCAGACGCUAAAUCUAC                                    CCCGU GAAGGUUCGUCCCCUUC                                                        AUGCCUCACCA    #12 (25)            GGCCAAACUGA                      GCAGACUCUAAAUUUGC                                    CACCA GAAGGUUCGCCCCCUUC                                                        AUGCCUCACCA    #13 (26)            GGCCAAACUGA                      GCAGACUC-AAAUCUGGC                                    CAUUC GAAGGUUCGCCCCCUUC                                                        AUGCCUCACCA    #14 (27)            GGCCAAACUGA                      GCAGACUCUAAAUC-GC                                    AGUGU GAAGGUUCGUGCCCUUC                                                        AUGCCUCACCA    #15 (28)            GGCCAAACUGA                      GCAGACUCUAAAUCAGC                                    GCGUG GAAGGUUCGUGCCCUUC                                                        AUGCCUCACCA    #16 (29)            GGCCAAACUGA                      GCAGACUCUAAAUCGGC                                    CGCAC GAAGGUUCGCCCCCUUC                                                        AUGCCUCACCA    #17 (30)            GGCCAAACUGA                      GCAGACACUAAAUUUGC                                    ACGAG GAAGGUUCGCCCCCUUC                                                        AUGCCUCACCA    #18 (31)            GGCCAAACUGA                      GCAGACCCUAAAUCUGC                                    CCCCG GAAGGUUCGUGCCCUUC                                                        AUGCCUCACCA    __________________________________________________________________________

E. Synthesis and Analysis of Individual EGS RNAs Based on SelectedChimeric Substrates

The best chimeric substrate selected was clone 9 whose corresponding RNAsequence is partially shown in sequence 9 (nucleotides 1 through 60 ofSequence ID No. 9) in Table 1. The clone 9 chimeric substrate wascleaved about 5.5 times more efficiently than the parent,non-randomized, chimeric substrate, mRNA-EGS^(CAT) chimera. Using thesequences of the selected chimeric substrates, nine individualcorresponding EGS RNAs were synthesized: EGS-1 (Sequence ID No. 17),EGS-4 (Sequence ID No. 19), EGS-5 (Sequence ID No. 20), EGS-6 (SequenceID No. 21), EGS-8 (Sequence ID No. 22), EGS-9 (nucleotides 1 through 60of Sequence ID No. 9), EGS-12 (Sequence ID No. 25), EGS-14 (Sequence IDNo. 27) and EGS-18 (Sequence ID No. 31), in order to probe the functionof these selected EGSs in directing RNase P to the target CAT mRNA.

Each of the individual EGS RNAs was mixed with ³² P-labeled CAT mRNA andthe mixtures were then exposed to RNase P. Every selected EGS RNAincreased the initial rate of the cleavage reaction, as measured duringthe linear phase of the reaction, over that with EGS^(CAT), and cleavageoccurred at the expected site in the target mRNA, as demonstrated byFIG. 5.

The most dramatic improvement in rates occurred with the EGS sequencebased on clone 9, EGS 9 (nucleotides 1 through 60 of Sequence ID No. 9),which directed cleavage of the CAT mRNA at an overall rate more than 30times faster than that observed with EGS^(CAT) in the complex. The threemost efficient EGSs tested, derived from clone 6, clone 8 and clone 9;all had a common sequence, UUCGUGC, in the T loop.

F. Secondary Structural Analysis of mRNA-EGS Complexes

The proposed secondary structure of the complex of CAT mRNA and EGS 9(Sequence ID No. 9) (FIG. 4B) can be compared with the parent CATmRNA-EGS^(CAT) complex (FIG. 4A). The structures of CAT mRNA-EGScomplexes were confirmed in part by partial digestion with RNases T1 andT2 under conditions that allowed formation of the mRNA-EGS complex andidentification of single-stranded regions in RNA. Partial digestion ofCAT mRNA-EGS complexes with RNases T1 and T2 (Pharmacia) were performedin RNase P assay buffer (50 mM Tris-HCl (pH 7.5), 10 MM MgCl₂, and 100mM NH₄ Cl). The reaction mixture contained substrate RNA labeled with ³²p! at its 5' terminus (2000 cpm), 0.2 mg/ml of rat 5S RNA as carrier,and three different concentrations, 2×10⁻⁴, 1×10⁻³ and 5×10⁻³ units/ml,of RNase T1 or RNase T2. Reactions were incubated at room temperaturefor 5 min. The samples were analyzed by PAGE in 12.5% sequencing gels.Double-stranded regions were identified by digestion with cobra venomnuclease (Pharmacia, Alameda, Calif.). Conditions for digestion withcobra venom nuclease were as described above for RNase T1 and T2 exceptthat incubation was at 37° C.

The sites of cleavage by the various nucleases in CAT mRNA-EGS^(CAT)complex are indicated by solid arrows in FIGS. 4A and 4D. The tRNAdomain in this structure is very similar to that found in a naturaltRNA. The results obtained with cobra venom nuclease indicate that thefirst few nucleotides in the analog of the D loop are involved in atertiary interaction, presumably with nucleotides in the variable loop(FIG. 4D). This interaction is either absent or much less extensive inthe complex with EGS 9 (Sequence ID No. 9) (FIG. 4E) and, indeed, thesame region in the analog of the D loop is susceptible to attack byRNases T1 and T2, an indication that it is in a single-strandedconformation (FIG. 4B). This result, together with the appearance of newsites of susceptibility to attack by cobra venom in the anticodon loopof EGS 9 confirm that this EGS endows the complex of the EGS and CATmRNA with new tertiary interactions that enhance the rate of cleavage byRNase P. The results of cleavages by nucleases also confirmed that theanticodon stem in EGS 9 was disrupted as a result of a single nucleotidedeletion. EGSs 9 and 14 are not similar to each other in terms of theirefficiency in directing RNase P to a target substrate. The onlydifference in their nucleotide sequences is in the variable loop, asshown by Table 1. This difference alone must account for the relativeinefficiency of EGS 14 in terms of targeting ability. Furthermore,digestion with RNase T1 of the complex that contained EGS 9 revealedstrong protection of the last nucleotide, G, in the variable loop fromattack by RNase T1, as shown by FIG. 4B. The role of this G may besimilar to that played by nucleotide 57 in tertiary interactions in tRNAmolecules; namely, the G may form hydrogen bonds with a nucleotide inthe CAT mRNA sequence to ensure folding of the "tRNA" domain of the mRNAsequence.

G. Anticodon Stem and Loop of an EGS Decrease Cleavage Rate of RNase Pand are Dispensable

An independent study of recognition of precursor tRNA substrates byhuman RNase P showed that the anticodon stem and loop form a dispensablestructural feature in the recognition of substrates by human RNase P. Todetermine whether the anticodon stem and loop may act in a negativefashion on the overall rate of cleavage of target. RNA by RNase P, twomore EGS RNAs were constructed, one being a deletion mutant that lackedthe equivalent of the anticodon stem and loop, EGS^(CAT) ΔAC (SequenceID No. 10), as compared to the parent EGS^(CAT) (FIG. 4A), and theother, EGS 19, being a derivative of EGS 9 in which the structure of theanticodon stem was restored.

DNA coding for EGS^(CAT) ΔAC was synthesized by PCR with pEGS^(CAT) DNA,described by Yuan et al., Proc. Nat. Acad. Sci. USA 89: 8006 (1992), astemplate. Oligonucleotide EC-1AAC (GCCAAACTG ACGTCATCGACTTCG, SequenceID No. 32) and M13 reverse primer (AACAGCTATGACCATG, Sequence ID No. 33)were used as primers. The DNA generated by PCR was digested with HindIIIand then inserted into pUC19 downstream from a T7 RNA polymerasepromoter sequence. EGS^(CAT) ΔAC RNA (Sequence ID No. 10) was preparedby transcription in vitro after the new plasmid DNA had been linearizedwith DraI. DNA that coded for EGS 19 was synthesized by PCR procedure ina manner similar to that used for the synthesis of DNA for EGS 9, witholigonucleotides SEC-1C (Sequence ID No. 15) and SEC-1I(GTAATACGACTCACTATAGGCCAAACTGAGCAGACTCTAAATCTGCAAACGGAAGGTTC, SequenceID No. 34): the newly inserted T residue in SEC-1I is underlined. TheDNA was transcribed in vitro with T7 RNA polymerase to give EGS 19 RNA.EGS 19 (Sequence ID No. 39) RNA differs from EGS 9 RNA only in theadditional U that restores the structure of the anticodon stem.

In EGS^(CAT) ΔAC (Sequence ID No. 10), the length of EGS^(CAT) wasreduced by 25%: the shorter deletion mutant directed cleavage of targetRNA about six times more efficiently than the parent EGS, as shown byFIG. 5. Restoration of the anticodon-stem structure, as in EGS 19,reduced the rate of cleavage of the target RNA with EGS 19 to four timeslower than that with EGS 9. These results, together with the measurementof the rates of reaction with EGSs selected in vitro, indicate asignificant inverse correlation between the efficiency of an EGS in thecleavage reaction and the existence of an anticodon stem in EGS RNA.

H. Stability of EGS-mRNA Complexes

The stability of EGS-mRNA complexes was measured based on both thebinding constants between the mRNA and each EGS and the dependence onMg²⁺ ions of the cleavage reaction, presuming that relatively highconcentrations of Mg²⁺ ions were needed to stabilize relatively unstablecomplexes. The dissociation constants (K_(d)) of mRNA-EGS complexes weremeasured directly by a gel mobility shift assay in polyacrylamide gelsthat contained 10 mM Mg²⁺ ions according to the method of Pyle et al.,Proc. Natl. Acad. Sci. USA 87: 8187-8191 (1990). A fragment of CAT mRNA,160 nucleotides in length, was prepared by transcription with T7 RNApolymerase in the presence of α-³² P!GTP. 10 μl EGS RNA in 2X bindingbuffer was heated at 80° C. for 4 min before it was mixed with an equalvolume of 2 nM CAT mRNA fragment in water. 1X binding buffer contains 50mM Tris-Cl (pH 7.5), 10 mM MgCI₂, 100 mM NH₄ Cl, 3% glycerol, 0.05%xylene cyanol. The mixtures were incubated at 37° C. for 20 min andimmediately separated on 5 % polyacrylamide gels at 9 watts. Theelectrophoresis buffer consisted of 36 mM Tris base, 64 mM HEPES, 0.1 mMEDTA, 10 mM MgCl₂ (pH 7.5 without any adjustment). Quantitation of freetarget RNA and of the complex was performed with a Betascope (Betagen,Waltham, Mass.). The free energies of binding were determined from theequation αG°=--RT 1n (1/K_(d)), where R=0.00198 kcallmol and T=310.15°K.

The dissociation constants for selected EGSs are shown in Table 2.

                  TABLE 2    ______________________________________    Dissociation constants of EGSs.                                  V.sub.max                    K.sub.d                           K.sub.m                                  (nmol/min) ×                                          V.sub.max /K.sub.m ×    Substrates      (nM)   (nM)   10.sup.-5                                          10.sup.-6    ______________________________________    pTyr                   10     2.9     2.90    CAT mRNA +      880    120    2.9     0.24    EGS.sup.CAT    (Sequence ID No. 8) +                    20     150    11.4    0.76    EGS.sup.CAT ΔAC    (Sequence ID No. 10) +                    210    125    16.3    1.30    EGS-5    (Sequence ID No. 20) +                    25     125    21.3    1.70    EGS-8    (Sequence ID No. 22) +                    78     125    30.0    2.40    EGS-9    (nucleotides 1-60 of Sequence                    710    130    12.5    0.96    ID No. 9) +    EGS-19    ______________________________________

Table 2 shows the kinetic parameters of EGS-directed cleavage of CATmRNA in vitro by RNase P from HeLa cells. K_(d) refers to measurementsof the dissociation constant for binding of EGS to CAT mRNA. The otherparameters were determined in standard assays of enzyme kinetics.V_(max) is the value obtained with 0.5 ml (0.6 units) of human RNase P.pTyr refers to the precursor to tRNA^(Tyr) from E. coli.

The results shown in Table 2 indicate that the K_(d) values of in vitroselected EGSs are 4 to 40 times lower than that of the parent EGS. Thus,the selected EGSs had higher affinity for the target RNA than didEGS^(CAT). The chimeric substrate derived from clone 9 was cleaved byRNase P at a rate only about 1.5 times faster than was the target in themRNA-EGS 9 complex, an indication that the ability of the EGS to bindtightly to the target RNA in solution must be a critical determinant inthe efficiency of the substrate complexes.

The differences in K_(d) values between complexes with EGS^(CAT)(Sequence ID No. 8) and the selected EGSs correspond to the contributionof -1 to -2.4 kcal/mol to the free energy of binding (αG°) with selectedEGSs (αG°) is -8.5 kcal/mol for the complex with EGS^(CAT), -10.1 forthe complex with EGS 9 (Sequence ID No. 9) and -10.9 for the complexwith EGS^(CAT) ΔAC (Sequence ID No. 10), thus revealing new interactionsin the selected EGS-mRNA complexes.

Deletion of the anticodon stem from EGS^(CAT) resulted in the K_(d) forEGS^(CAT) αAC being 44 times lower, and restoration of the stem of EGS 9(EGS 19) resulted in a K_(d) that was ten times higher than that for EGS9 and was close to that of EGS^(CAT) (Table 2). Thus, the intactanticodon stem stabilized a conformation of the EGS that could bind asstrongly to the target RNA as does an EGS with no organized anticodonstem. Accordingly, the enhancement of the ability of the selected EGSsfor targeting RNA can be assigned, in part, to the increase in thestrength of their binding to target RNA.

Cleavage of mRNA directed by the original EGS^(CAT) requires Mg²⁺ ionswith an optimum concentration of 25 mM. By contrast, the reaction withcertain selected EGSs, namely, EGS 6 (Sequence ID No. 21) and EGS 9(nucleotides 1 through 60 of Sequence ID No. 9), proceeds optimally in 2to 10 mM Mg²⁺ ions. This latter concentration is close to the optimalconcentration of Mg²⁺ ions for processing of tRNA precursors by humanRNase P, reported by Doersen et al., J. Biol. Chem. 260: 5942 (1985).Since high concentrations of Mg²⁺ ions are especially effective in theneutralization of repulsion between adjacent regions of the phosphatebackbone and the stabilization of RNA folding, the results indicate thatthe selected EGSs can achieve the appropriate folded structures in thecomplex with target RNAs without the aid of high concentrations of Mg²⁺ions.

A kinetic analysis was performed to determine the Michaelis constant(K_(m)) and the maximum velocity (V_(max)) of the enzymatic reactions.The cleavage of the precursor to tRNA^(Tyr) from E. coli and of CAT mRNAin mRNA-EGS complexes was assayed at various substrate concentrationsboth above and below the K_(m) for these substrates. Aliquots werewithdrawn from reaction mixtures at regular intervals and analyzed onpolyacrylamide-urea gels. Values of K_(m) and V_(max) were obtained fromLineweaver-Burk double-reciprocal plots. The effective concentrations ofsubstrate used were calculated as the concentration of the complex oftarget mRNA with EGS as determined from the K_(d) values shown in Table2. The K_(m) for the precursor to tRNA^(Tyr) (pTyr) is 10 nM with humanRNase P, whereas the K_(m) value of the complex of mRNA-EGS^(CAT) istwelve-fold higher (Table 2). The K_(m) for all the selected EGSs testedwas the same as that of EGS^(CAT). However, the maximum velocities ofthe reactions with selected EGSs were up to ten times higher than thatwith the original EGS. Thus, the value of V_(max) /K_(m) for selectedEGSs was increased. The value of V_(max) /K_(m) of EGS 9, was forexample, ten-fold higher than that of EGS^(CAT) and was very close tothat of the tRNA^(Tyr) precursor. When the anticodon stem of EGS 9 wasrestored, however, as it was in EGS 19, V_(max) fell about 2.5-fold,suggesting that the rate of release of the product was specificallyreduced by physical interactions of the enzyme with an intact anticodonstem. These data show that the enhanced abilities for targeting of theselected EGSs, as measured in the overall rate of the cleavage reaction,were due to both enhanced affinity of binding to substrate RNAs and toincreases in the velocity of the enzymatic reaction.

EXAMPLE 6 Inhibition of Viral mRNA with Human Ribonuclease P

Herpes simplex virus was used to demonstrate that EGS can be used totarget a viral gene in vivo to inhibit viral replication. Herpes simplexviruses are DNA-containing viruses that infect cells, induce synthesisof messenger RNAs, which are transcribed to produce enzymes related toDNA synthesis and breakdown: including thymidine kinase, DNA polymeraseand a DNA exonuclease, and viral DNA and viral structural proteins aremade and assembled into infectious viral particles. The structure andorganization of the herpes simplex virus genome is known, for example,as reported by Roizman, Cell 16: 481-494 (1979). The nucleotide sequenceof the thymidine kinase gene of herpes simplex type 1 was described byWagner et al., Proc. Natl. Acad. Sci. USA 78: 1441-1445 (1981).

An EGS, shown in FIG. 6A, was designed to target the mRNA encodingthymidine kinase (TK). The target site for RNase P cleavage is about 25nucleotides downstream from the TK translation initiation site. An EGSforming an approximately three-fourths-like tRNA was designed and shownin vitro to cleave the TK sequence at the proposed cleavage site. Twoother EGSs, which contain a single point mutation in the T-loop (C to G)or deletion of the anti-codon region, shown in FIGS. 6B and 6C,respectively, were constructed based on the results observed in Example5.

Cell lines and EGS expression vectors were then constructed. Five celllines were constructed by transfecting plasmid DNAs into human143TK-cells, which can be obtained from the American Type CultureCollection, Rockville, Md.

Plasmid pFL116 was constructed using pGEM-7Z (Promega, Wis.) andincorporating a gene for neomycin resistance (Neo), which iscommercially available from Clontech or Stratagene, Calif. EGS DNA(FIGS. 6A, 6B, and 6C) was digested with KpnI and inserted into theplasmid pmU6(-315/1), described by Yuan et al., Proc. Natl. Acad. Sci.USA 89: 8006-8010 (1992) and Das et al., EMBO 7: 503-512 (1988), at thePstI (blunted)/KpnI site. This plasmid contains the promoter for thegene for U6 small nuclear RNA, a very strong promoter, and a signal fortermination of transcription (T cluster) by RNA polymerase II. EGSplasmids were designated pFL104, pFL109, and pFL112, respectively. Theplasmid based on sequence 9 in Table 1 was used as a control.

The cells were stably transfected using a calcium phosphateprecipitation method described by Wigler et al., Proc. Natl. Acad. Sci.USA 76: 1373-1376 (1979), with the pFL116 to yield CL116, plasmids EGS 9and pFL116 to yield CLCAT, plasmids pFL104 and pFL116 to yield CL104,plasmids pFL109 and pFL116 to yield CL109, and plasmids pFL112 andpFL116 to yield CL112, followed by neomycin selection. Cells werecloned, expanded and RNA isolated. Both total and cytoplasmic RNA wasisolated and the RNase protection method described in "MolecularCloning: A Laboratory Manual, Second Edition" Sambrook et al., (ColdSpring Harbor Laboratory Press, 1989) at pages 7.71-7.78. This is anextremely sensitive assay where digestion of RNA:RNA hybrids formedusing radiolabelled probe is used to assess which cell clones expressthe EGS.

The results indicated that the EGSs are expressed in both nuclei andcytoplasm.

Cells were then infected with herpes simplex virus using a multiplicityof infection (MOI) of 1 to 1.5, specifically, 1 to 1.5 million viralparticles/i million cells, in order to resemble a natural infection withvirus. RNA was harvested at 4, 8 and 12 hours post-infection. Theinternal control probe was used to detect the mRNA levels of HSV α47 andlate genes U₅ 10 and U₅ 11. The probe is selected to assure thedetection of a high level of viral mRNA expression over the entire cycleof viral infection.

The results are shown in FIG. 7. TK mRNA expressed was decreased 0% inthe control CL-CAT, 30% in CL-109, 45% in CL-104, and 65% in CL-112.

EXAMPLE 7 RNase P Internal Guide Sequences

A. Construction of plasmids, catalytic RNAs and RNA substrates forstudies in vitro

DNA templates for transcription in vitro of RNA substrates tk7, tk46,and cat7 were constructed by annealing the T7 promoter containingoligonucleotide, OliT7 (5'-TAATACGACTCACTATAG-3') (Sequence ID No. 41)with oligo-nucleotides Olitk7 (5'-CGCAGAC GGTCCTATAGTGAGTCGTATTA-3')(Sequence ID No. 42), OliTK46 (5'-ACCGCCGCAGCCTGGTCGAACGCAGACGCGTGTTGATGGCAG GGGTCTATAGTGAGTCGTATTA-3')(Sequence ID No. 43), and Olicat7 (5-ATGCCTCGGTCCTATAGTGAGTCGTATTA-3')(Sequence ID No. 44), respectively. Plasmids pTK117 and pTK146 arederivatives of pUC19 and were described by Guerrier-Takada and Altman,Proc. Natl. Acad. Sci. USA 89: 1266-1270 (1992). The DNA sequencescoding for M1 RNA (pTK117) and mutant M1 RNA with a deletion fromnucleotides 167 to 377 (pTK146), are under the control of the T7 RNApolymerase promoter. The DNA templates for M1TK19, M1TK16, M1TK13,M1TK10, M1TK5, and M1CAT13 were constructed by the polymerase chainreaction (PCR) with the gene for M1 RNA as found in plasmid pTK117 withOliT7 as the 5'primer oligonucleotide and 3' primers that contained theappropriate guide sequences. The 3' primers were OliTK19(5'-GTGGTGTCTGCGTTCGACCAGGCTATGAC CATG-3') (Sequence ID No. 45), OliTK16(5'GTGGTGTCTGCGTTC GACCAGTATGACCATG-3') (Sequence ID No. 46), OliTK13(5'-GTGGTGTCTGCGTTCTATGACCATG-3') (Sequence ID No. 47), OliTK10(5'-GTGGTGTCTGCGTTCTATGACCATG-3') (Sequence ID No. 48), OliTK5(5'-GTGGTGTCTGTATGACCATG-3') (Sequence ID No. 49), and OliCAT13(5'-GTGGTGAGGCATTTCAGTTATGAC CATG-3') (Sequence ID No. 50). The 3'proximal sequences of 10 no serve as the primers for the PCR with thepUC19 sequence. The underlined sequences and the bold sequencescorrespond to the 3° CCAC sequence and the guide sequences,respectively. The DNA template for ΔM1(167-377)TK13 RNA was constructedby PCR with the sequence for M1 RNA in pTK146 and primers OliT7 andOliTK13. The DNA template for EGS TK16 was constructed by PCR with the5' primer OliT7 and the 3' primer OliTK16 for DNA template pUCT7, whichwas derived from pUC19 by insertion of a T7 promoter sequence into theBamHI site. The CAT mRNA fragment of 550 nt was synthesized with T7 RNApolymerase and EcoRI-digested pCAT-1 plasmid DNA (Promega Inc.) whilethe TK mRNA fragment of 450 nt was synthesized from plasmid pTK101 DNAin which the sequence for TK mRNA is under control of the T7 promoter.

B. Assays for cleavage by M1GS RNA.

RNA enzyme (20 nM) and substrate (50 nM), either uniformly labeled withα-³² P!GTP or 5' end-labeled with γ-³² P!ATP, were incubated for 30 minsat 37° C. or 50° C. in buffer A (50 mM Tris, pH 7.5, 100 mM NH₄ Cl, 100MM MgCl₂) or buffer B (50 mM Tris, pH 7.5, 100 mM NH₄ Cl) that containedMgCl₂ at various concentrations. Reactions were stopped by the additionof 8M urea and the cleavage products were then separated on either 15%or 20% polyacrylamide gels that contained 8M urea. C5 protein and humanRNase P protein were purified from E. coli and HeLa cells, respectively,as described previously by Vioque et al., J. Mol. Biol. 202: 835-848(1988), and Bartkiewicz et al., Genes & Dev. 3: 488499 (1989). The RNaseP holoenzyme from E. coli was assembled by mixing M1 RNA and C5 proteinat a molar ratio of 1:20.

Assays to determine kinetic parameters under single- andmultiple-turnover conditions were performed as described previously byGuerrier-Takada et al., Cell 35: 849-857 (1983), and Liu and Altman,Cell 77: 1083-1100 (1994). Cleavage was assayed at variousconcentrations of substrate, in 2- to 20-fold excess over enzymeconcentration, both above and below the K_(m) for the substrate.Aliquots were withdrawn from the reaction mixtures at regular intervalsand the cleavage products were separated in polyacrylamide-urea gels.quantitation was carried out with a phosphorinager (Molecular Dynamics).The values of K_(m) and k_(cat) were obtained from Lineweaver-Burkdouble reciprocal plots. In single turnover experiments, trace amountsof substrates were used and the concentrations, 1 nM, were much lowerthan the K_(m) (>80 nM). The concentration of enzyme ranged from 5 nM to200 nM. The observed rate of cleavage (k_(obs)) was determined, and thevalue of k_(cat) /K_(m) was obtained from the equation k_(cat) /K_(m)=k_(obs) / E!, where E! is the concentration of the enzyme.

C. Viruses, cells and antibodies.

The properties of HSV-1(F), a prototype of human herpes simplex virus 1have been described by Ejercito et al., J. Gen. Virol. 2: 357-364(1968). The retroviral vector LXSN, retroviral packaging cell lines,PA317 (amphotropic) and ψACRE (ectopic), and their maintenance andpropagation were described by Miller and Rosman, BioTechniques 7:980-990 (1989), and Danos and Mulligan, Proc. Natl. Acad. Sci. USA 85:6460-6464 (1988). The rabbit polyclonal antibody against thymidinekinase of HSV-1 (F) was described by Liu and Summers, Virology 163:638-642 (1988), and the mouse monoclonal antibody MCA406 against HSV-1ICP35 protein was described by Liu and Roizman, J. Virol. 65: 206-212(1991), and purchased from Harlan Bioproducts for Sciences Inc.(Indianapolis, Ind.). The secondary antibodies used in the Westernblots, anti-rabbit or anti-mouse IgGs conjugated with horseradishperoxidase, were purchased from Vector Laboratories Inc. and Bio-RadInc., respectively.

D. Construction of plasmids for studies in vivo

The promoter sequence for U6 small nuclear RNA and a signal for thetermination of transcription (T cluster) by RNA polymerase III,described by Yuan et al. (1992), were inserted into the EcoRI site forthe retroviral vector LXSN, described by Miller and Rosman (1989), tocreate pRVO. Retroviral constructs PB2, M1TK and ΔM1TK were constructedby placing the DNA sequence that coded for the EGS with the ability totarget the mRNA for influenza viral protein PB2, m1TK13 RNA andΔM1(167-377)TK13 RNA under the control of the U6 promoter in plasmidpRVO, respectively. Plasmid pTK129 and pTK141 were constructed,respectively, by placing the BglII-MluI fragment (87 nt) of the HSV-1(F)BamHI Q fragment and BamHI-AccI fragment (181 nt) of the HSV-1 (F) BamHIZ fragment under the control of a phage T3 RNA polymerase promoter.These two fragments correspond to the sequences that encode the 5'sequence of TK mRNA and of the overlapping transcripts for the HSV-1α47, Us10, and Us11 genes, respectively, as described by McMeoch et al.,J. Gen. Virol. 69: 1531-1574 (1988).

E. Construction of cell lines

The protocols were modified from Miller and Rosman (1989). In brief,cells were transfected with retroviral vector DNAs with the aid of amammalian transfection kit purchased from Stratagene Inc. (La Jolla,Calif.). Forty-eight hours post transfection, neomycin (Gibco-BRL) wasadded to the culture medium at a final concentration of 600 μg/ml. Cellswere subsequently selected in the presence of neomycin for two weeks andneomycin resistant cells were cloned and allowed to proliferate inneomycin-containing medium, as described by Sambrook et al. (1989). Thenewly constructed cell lines, NB2, ≢M1TK, and M1LTK, and a control cellline in which cells were transfected with LXSN vector DNA, wereindistinguishable in terms of cell growth and viability for up to twomonths. Finally, aliquots of these cells were either frozen forlong-term storage in liquid nitrogen or used immediately for furtherstudies in vivo.

F. Viral infection and preparation of RNA and protein extracts

Approximately 10⁶ cells in a T25 flask were either mock-infected orinfected with HSV-1 in 1.5 ml of Medium 199 (M199; GIBCO) supplementedwith 1% fetal calf serum. After 2 hours of exposure of cells to virus at37° C., the medium was replaced with Dulbecco's modified Eagle's mediumsupplemented with 5% fetal bovine serum. The cells were incubated for 11hours before harvesting for isolation of viral mRNA and/or protein. RNAand protein extracts were prepared from cells that had either beenmock-infected with HSV-1, as described previously by Jenkins and Howett,J. Virol. 52: 99-107 (1984), and Liu and Roizman (1991).

G. RNase protection assay for viral mRNA expression

The RNA probes used to detect TK mRNA and the transcripts of the α47,Us10, and Us11 genes were synthesized in vitro with T3 RNA polymerase(Promega, Madison, Wis.) from the DNA templates pTK129 and pTK141,respectively, that had been linearized with EagI. RNase protectionassays were performed as described previously by Yuan et al. (1992). Theprotected RNA products were separated in 8M urea 8% polyacrylamide gelsand quantitated with a phosphorimager. quantitation was performed in thelinear range of RNA detection.

H. Electrophoretic separation and staining with antibodies ofpolypeptides from infected cells

Denatured, solubilized polypeptides from cell lysates were separated onSDS-9% v/v! polyacrylamide gels. The separated polypeptides weretransferred electrically to nitrocellulose membranes and allowed toreact in an enzyme-linked immunoassay with antibodies against eithermouse or rabbit IgG that had been conjugated with horseradish peroxidaseafter reaction with antibodies against HSV-1 TK or ICP35. The membraneswere subsequently stained with the color-development substrate fromperoxidase substrate kit purchased from Vector Laboratories Inc. orreacted with the chemiluminescent substrate in a LumiGLO™chemiluminescence kit (Kirkegaard and Perry Laboratories Inc.) andsubsequently subjected to exposure to X-ray film. Finally, the amountsof TK and ICP35 protein on the membrane were quantitated by scanning thefilms with a densitometer (Bio-Rad, Inc.). Quantitation was performed inthe linear range of protein detection.

I. Cleavage of model substrates by M1GS RNA in vitro

The HSV-1 TK gene has been well characterized and is reviewed by Roizmanand Sears, "Virology", 2nd edition, edited by Fields et al., pages1795-1841 (Raven Press, New York, 1990). Although the TK gene product isnot essential for viral replication in tissue culture cells, because itis so well studied we have used it as a model target for geneinactivation by M1GS RNA. DNA encoding a guide sequence (TK 13) thatcontains a sequence of 13 nt complementary to the 5' terminal sequenceof mRNA for HSV-1 TK protein was covalently linked to the 3' end of DNAthat encoded M1 RNA (FIG. 9). The RNA transcript of this construct,M1TK13, cleaved target RNA, tk7, that contains 7 nt of the 5' sequenceof TK mRNA and an unrelated sequence of 5 nt that serves as a leadersequence (Sequence ID No. 52). Cleavage in the target occurs at position5, yielding two cleavage products of 5 nt and 7 nt in length,respectively. M1TK13 remains unchanged during the reaction, as expectedof a true enzyme, since only labeled, full-length M1TK13 RNA is detectedafter incubation of the reaction mixture. Attachment of guide sequencesto the 3' end, rather than to the 5' end, of M1 RNA in the constructionof M1GS RNA is preferred because the presence of a 3' terminal CCAsequence in the guide sequence is important for maximum efficiency ofcleavage. Furthermore, additional sequences downstream from the CCAsequence lower the rate of cleavage of substrates.

M1GS RNA only cleaves substrates that are complementary to the guidesequence. M1TK13 RNA can cleave substrate tk7 but not substrate cat7,which contains a sequence of 7 nt from the mRNA for chloramphenicolacetyltransferase (CAT). However, M1CAT13 RNA, in which the guidesequence contains a sequence of 13 nt complementary to CAT mRNA, canefficiently cleave cat7 but not tk7. Therefore, M1GS RNA appears to actas a sequence-specific endonuclease, recognizing its substrates throughspecific base-pairing between the GS and the target sequence, as hasalso been shown by Frank et al., Biochemistry 33: 10800-10808 (1994),with different constructs. To determine whether longer RNAs can bespecifically cleaved by these new RNA enzymes, uniformly ³² P!-labeledfragments of the TK (450 nts) and CAT (550 nts) mRNA sequences (FIG. 10)were incubated with either M1TK13 RNA or M1CAT13 RNA. Sequence-specificand efficient cleavage of these RNA substrates by the appropriate RNAenzymes resulted.

A set of RNA enzymes, designated M1TK19, M1TK16, M1TK13, M1TK10 andM1TK5, were constructed in which the GSs contained sequences of 19, 16,13, 10, 5 nucleotides complementary to the TK mRNA sequence,respectively (FIG. 11), in order to relate the length of the targethelix to efficiency of cleavage. The substrate tk46, which contains a TKmRNA sequence of 46 nt, was cleaved by all the constructs and thecleavage site was determined to be at position G21, as expected (FIG.11). Further kinetic analysis of cleavage of substrate tk46 by M1TK16,M1TK13, M1TK10, and M1TK5 RNAs was also performed and the results areshown in Table 3.

                  TABLE 3    ______________________________________    Kinetic parameters of reactions catalyzed by    various M1GS RNA constructs    Enzyme    K.sub.2 (μM)                       k.sub.cat (min.sup.-1)                                   k.sub.cat /K.sub.m                                         k.sub.cat /K.sub.m *    ______________________________________    M1TK16    0.08     0.14        1.6   2.4    M1TK13    0.09     0.21        2.3   2.4    M1TK10    0.15     0.10        0.7   0.7    M1TK5     0.22     0.03        0.1   0.1    ______________________________________

Values of k_(cat) increased in the rank order from M1TK5 to M1TK16 RNA.Values of K_(m) decreased progressively with increasing length of theduplex. The overall K_(m) of the reaction undoubtedly involves both thesubstrate binding to the guide sequence (helix formation) and docking ofthe double helical segment in the active site of M1 RNA, a complexprocess. There was one exception to the rule of increasing k_(cat) withlength of guide sequence: k_(cat) decreased from M1TK13 to M1TK16. Thislast result can be explained if release of product becomes therate-limiting step in the reaction governed by M1TK16 RNA under single-and multiple-turnover conditions as described by Fersht, "Enzymestructure and mechanism", 2nd edition (W. H. Freeman and Co., New York,1985). Further analysis of the reaction with M1TK13 showed that about 5pmol of tk46 were cleaved by 1 pmol of M1TK13 in 30 minutes, indicatingthat the RNA enzyme turns over 5 times during the incubation period.This result is consistent with the value of k_(cat) measured byclassical Michaelis-Menten kinetic analysis (see Table 3).

M1GS RNAs act more efficiently than does M1 RNA in the classic cleavagereaction in trans. M1TK16 cleaves tk46 at least 10 times faster thandoes M1 RNA when using a separate guide sequence, TK16, under equivalentexperimental conditions. Moreover, M1GS RNA cleaves its substrates moreefficiently in buffers that more closely mimic physiological conditionsin terms of magnesium ion concentration (10 mM MgCl₂) than does M1 RNAwith a separated guide sequence in buffers that contain 100 MM MgCl₂, anindication that the high concentrations of Mg²⁺ ions are needed tomediate binding of the substrate to M1 RNA alone, as indicated byKazakov and Altman, Proc. Natl. Acad. Sci. USA 88: 9193-9197 (1991), andSmith and Pace, Biochemistry 32: 5273-5281 (1993). Further analysis ofthe cleavage reaction of M1GS RNA indicated that the cleavage proceedsoptimally at a temperature of 62° C., at concentrations of monovalentcations of 100 mM, and at concentrations of Mg²⁺ ions of 60-100 mM.

To prove that the catalytic activity of M1GS RNA resides in the sequencethat encodes M1 RNA, a set of M1TK RNAs was constructed in which eachRNA had a deletion in a different region of the M1 RNA sequence but inwhich each had the same guide sequence, TK13. The various M1 RNAdeletion mutants, for example, Δ167-377, Δ1-163, and Δ65, lack thecatalytic activity needed to process pre-tRNAs, as shown byGuerrier-Takada et al., Science 286: 1578-1584 (1989) andGuerrier-Takada and Altman (1992). M1GS RNA constructs with thesedeletion mutants and the linked TK13 sequence were unable to cleavetk46.

Stimulation of the activity of M1GS RNA by proteins

C5 protein, the protein subunit of RNase P from E. coli, increases therate of cleavage of natural substrates by M1 RNA as described byGuerrier-Takada et al. (1983) and Reich et al., Science 239: 178-181(1988). C5 protein also stimulates the cleavage by M1GS RNA by a factorof 30 or more. Furthermore, cleavage by M1GS RNA can be stimulated atleast five-fold by a partially purified preparation of human RNase P.This enhancement in rate was anticipated as it had been previously shownthat protein from a crude preparation of human (HeLa cells) RNase P canenhance the cleavage of ptRNA by M1 RNA as described by Gold and Altman,Cell 44: 243-249 (1986). The rate stimulation cannot be ascribed toresidual human RNase P activity as it, alone, cannot cleave substratetk46 in complexes with catalytically inactive ΔM1(167-377)TK13 RNA. Thelast result is consistent with observations that a simple stem-loopstructure can serve as a substrate for M1 RNA but not for eukaryoticRNase P. This was shown specifically with human and X. laevis RNase P byYuan and Altman, Science 263: 1269-1273 (1994), and Carrara et al.,Proc. Natl. Acad. Sci. USA. No stimulation of cleavage of substrate tk46by M1TK13 RNA was observed when fractions devoid of human RNase Pactivity were used. Accordingly, it is expected that when M1GS RNAconstructs are present in mammalian cells, their activity should beenhanced by endogenous proteins.

J. Expression in vivo of M1GS RNA.

To express M1GS RNA in mammalian cells, retroviral constructs, M1TK andΔM1TK, were generated by cloning genes for M1TK13 and ΔM1(163-377)TK13into the retroviral vector LXSN under the control of a mouse U6 snRNApromoter, described by Das et al., EMBO J. 7: 503-512 (1988), and Yuanet al. (1992) (FIG. 12). An additional retroviral DNA construct, NB2,containing the U6 promoter and an external guide sequence (NB2) thattargets the mRNA that encodes the PB2 protein of human influenza virus,was used as a control.

The targeted cleavage site of TK mRNA expressed in cells infected withHSV-1 appears to be modifiable by dimethyl sulfate (DMS) in vivo, basedon Peattie and Gilbert, Proc. Natl. Acad. Sci. USA 77: 4679-4682 (1980),Inoue and Cech, Proc. Natl. Acad. Sci. USA 82: 648-652 (1985), Climieand Friesen, J. Biol. Chem. 263: 15166-15175 (1988), and Ares and Igel,Genes & Dev. 4: 2132-2145 (1990). This is an indication that this sitemight be accessible for binding to M1GS in vivo. Amphotropic packagingcells (PA317) were transfected with retroviral vector DNAs to produceretroviruses that encoded the genes for M1GS RNA. Subsequently,esotropic packaging cells (ψCRE) were infected with these retroviruses,and cells expressing the retroviruses and ribozymes were cloned. Stableexpression of M1GS RNAs was demonstrated by an RNA protection assay witha probe that was complementary to the sequence of M1 RNA and RNAisolated from these cell lines. Furthermore, RNA extracted fromM1TK-expressing cells can cleave substrate tk46 in vitro while RNAextracted from the parental ψCRE cells, cells that expressed NB2 RNA andψM1TK RNA do not show cleavage activity. These results demonstrate thatthe M1TK RNA that is expressed in cultured cells was intact andcatalytically active.

K. Inhibition of expression of TK of HSV-1 in cells that express M1GSRNA

Cells were infected with herpes simplex virus 1 (HSV-1) at amultiplicity of infection (MOI) of 0.05 to 0.1. Levels of TK mRNA in theinfected cells were determined by an RNase protection assay with an RNAprobe (TK probe) that contained a sequence of 87 nt complementary to the5' proximal sequence of TK mRNA. An RNA probe (α47), containing asequence of 181 nt complementary to the overlapping regions of α47,Us10, and Us11 mRNAs encoded by HSV-1, was used to determine the levelsof these latter mRNAs. The levels of the latter RNAs were used asinternal controls for quantitation of expression of TK mRNA. FIG. 13graphically summarizes the results of the RNase protection experimentswith both the TK and α47 probes. A reduction of about 80±5%, averagedover four experiments, in the level of TK mRNA expression was observedin cells that expressed M1TK RNA while cells that expressed αM1TK RNAonly exhibited a reduction of 9±3%, averaged over four experiments.Thus, it appears that cleavage of TK mRNA by M1TK RNA did indeed occurinside cells, with a subsequent reduction in the level of TK mRNA thatcould be translated. No products of the cleavage of TK mRNA weredetected in our RNase protection assays presumably because these RNAs,which lack either a cap structure or a polyA sequence, are rapidlydegraded by intracellular RNases.

Protein extracts of the infected cells were analyzed for the presence ofthe TK polypeptide. Polypeptides were transferred to two identicalmembranes and one was stained with a TK-specific antibody (anti-TK),described by Liu and Summers (1988), while the other was stained with amonoclonal antibody against the capsid protein ICP35 of HSV-1(anti-ICP35), described by Liu and Roizman (1991). The expression ofICP35 serves as an internal control for the quantitation of expressionof TK. The results of four independent experiments are summarized inFIG. 13: a reduction of at least 76±5%, averaged over four experiments,in the level of TK protein was observed in cells that expressed M1LTKRNA while a reduction of only 10±4%, averaged over four experiments, wasseen in cells that expressed ΔM1TK RNA. The low level of inhibitionfound in cells that expressed ≢M1TK RNA was presumably due to anantisense effect.

These examples show that when M1 RNA is converted to an RIGS (M1GS RNA),it cleaves one particular substrate in a reaction governed by sequencespecificity. A custom-designed RIGS specific for thymidine kinasesequence (M1TK RNA), cleaves the TK mRNA in vitro and can be stablyexpressed in mammalian cells and can reduce the level of expression ofTK by≧75% when these cells are infected with HSV-1. The reduction in thelevel of expression of TK that can be ascribed to the antisense effectof the (internal) guide sequence is no more than 15% of the totalinhibitory effect. Moreover, the high degree of sequence specificity,which is governed by a guide sequence that hybridizes to a complementarysequence in the substrate, makes our construct suitable for use as atool for targeting RNAs in vivo. The optimal length of an antisensesequence for in vivo targeting is about 13 nucleotides, as discussed byStein and Cheng, Science 261: 1004-1012 (1993). The extent ofinactivation that we observed was very similar to that achieved whenendogenous RNase P is used as the catalytic agent to cleave complexes oftarget TK mRNA and separate EGSs expressed from synthetic genes thathave been stably incorporated into human cells in tissue culture.

The activity of M1GS RNA was stimulated in vitro by C5 protein andmammalian proteins. Gold and Altman (1986) suggested that C5 protein andprotein subunits of human RNase P might bind to homologous sequences andsimilar structures that are found in both M1 RNA and H1 RNA, the RNAcomponent of RNase P from HeLa cells. The cleavage reactions by M1LGSRNA in cells that contain proteins that bind RNase P catalytic RNA areexpected to proceed at rates higher than those observed in vitro. Inparticular, higher rates are expected in the presence of additional,non-specific, RNA-binding proteins, RNA chaperons, which are known tostimulate the activity of other RNA enzymes as indicated by Tsuchihashiet al., Science 262: 99-102 (1993), Coetzee et al., Genes & Dev. 8:1575-1588 (1994), Bertrand and Rossi, EMBO J. 13: 2904-2912 (1994), andHerschlag et al., EMBO J. 13: 2913-2924 (1994).

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 65    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 88 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    GAACAUUUUGAGGCAUUUCAGUCAGUUGGCCAAACUGAGCAGACUCUAAA50    UCUGCNNNNNGAAGGUUCNNNNCCUUCAUGCCUCACCA88    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 61 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    TAATACGACTCACTATAGAACATTTTGAGGCATTTCAGTCAGTTGGCCAA50    ACTGAGCAGAC61    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 61 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    TGGTGAGGCATGAAGGNNNNGAACCTTCNNNNNGCAGATTTAGAGTCTGC50    TCAGTTTGGCC61    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 99 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    GAAUACACGGAAUUGGUGGGGUUCCCGAGCGGCCAAAGGGAGCAGACUCU50    AAAUCUGCCGUCAUCGACUUCGAAGGUUCGAAUCCUUCCCCCACUGCCA99    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 71 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    GCCAAAGGGAGCAGACUCUAAAUCUGCCGUCAUCGACUUCGAAGGUUCGA50    AUCCUUCCCCCACCACCAUCA71    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    GAAUACACGGAAUUGGUGGGGUUCCCGA28    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: mRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    AUGGAACAUUUUGAGGCAUUUCAGUCAGUUUAA33    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 70 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    GGCCAAACUGAGCAGACUCUAAAUCUGCCGUCAUCGACUUCGAAGGUUCG50    AAUCCUUCAUGCCUCACCAU70    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 61 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    GGCCAAACUGAGCAGACUCUAAAUCGCAAACGGAAGGUUCGUGCCCUUCA50    UGCCUCACCAU61    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 53 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    GGCCAAACUGACGUCAUCGACUUCGAAGGUUCGAAUCCUUCAUGCCUCAC50    CAU53    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 341 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Homo sapiens    (x) PUBLICATION INFORMATION:    (A) AUTHORS: Altman et al.    (C) JOURNAL: Genomics    (D) VOLUME: 18    (F) PAGES: 418-422    (G) DATE: 1993    (K) RELEVANT RESIDUES IN SEQ ID NO:1: FROM 1 TO 341    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    AUAGGGCGGAGGGAAGCUCAUCAGUGGGGCCACGAGCUGAGUGCGUCCUG50    UCACUCCACUCCCAUGUCCCUUGGGAAGGUCUGAGACUAGGGCCAGAGGC100    GGCCCUAACAGGGCUCUCCCUGAGCUUCGGGGAGGUGAGUUCCCAGAGAA150    CGGGGCUCCGCGCGAGGUCAGACUGGGCAGGAGAUGCCGUGGACCCCGCC200    CUUCGGGGAGGGGCCCGGCGGAUGCCUCCUUUGCCGGAGCUUGGAACAGA250    CUCACGGCCAGCGAAGUGAGUUCAAUGGCUGAGGUGAGGUACCCCGCAGG300    GGACCUCAUAACCCAAUUCAGACUACUCUCCUCCGCCCAUU341    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    GCCAAACTGAGCAGACTC18    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    GCGCGGTACCAAAAATGGTGAGGCATGAAGG31    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    GGCCGTAATATCCAGCTGAACGG23    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    TGGTGAGGCATGAAGG16    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    TAATACGACTCACTATAGGCCAACTGAGCAGAC33    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 61 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    GGCCAAACUGAGCAGACUCUAAAUCGGCCCUUCGAAGGUUCGCCCCCUUC50    AUGCCUCACCA61    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 62 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    GGCCAAACUGAGCAGACUCUAAAUCUGCACGAGAGAAGGUUCGUGCCCUU50    CAUGCCUCACCA62    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 61 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    GGCCAAACUGAGCAGACUCUAAACUGGCCUAACGAAGGUUCGCCCCCUUC50    AUGCCUCACCA61    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 60 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    GGCCAAACUGAGCAGACUCUAAAUUGCCCAACGAAGGUUCACCCCCUUCA50    UGCCUCACCA60    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 59 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    GGCCAAACUGAGCAGACUCCAAAUCCACCAAGAAGGUUCGUGCCCUUCAU50    GCCUCACCA59    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 60 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    GGCCAAACUGAGCAGACUCUAAACUCCUCCCAGAAGGUUCGUGCCCUUCA50    UGCCUCACCA60    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 61 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    GGCCAAACUGAGCAGACUCUAAAUCGGCCUACGGAAGGUUCGCCCCCUUC50    AUGCCUCACCA61    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 61 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    GGCCAAACUGAGCAGACGCUAAAUCUACCCCGUGAAGGUUCGUCCCCUUC50    AUGCCUCACCA61    (2) INFORMATION FOR SEQ ID NO:25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 61 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    GGCCAAACUGAGCAGACUCUAAAUUUGCCACCAGAAGGUUCGCCCCCUUC50    AUGCCUCACCA61    (2) INFORMATION FOR SEQ ID NO:26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 61 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    GGCCAAACUGAGCAGACUCAAAUCUGGCCAUUCGAAGGUUCGCCCCCUUC50    AUGCCUCACCA61    (2) INFORMATION FOR SEQ ID NO:27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 60 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    GGCCAAACUGAGCAGACUCUAAAUCGCAGUGUGAAGGUUCGUGCCCUUCA50    UGCCUCACCA60    (2) INFORMATION FOR SEQ ID NO:28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 61 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    GGCCAAACUGAGCAGACUCUAAAUCAGCGCGUGGAAGGUUCGUGCCCUUC50    AUGCCUCACCA61    (2) INFORMATION FOR SEQ ID NO:29:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 61 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    GGCCAAACUGAGCAGACUCUAAAUCGGCCGCACGAAGGUUCGCCCCCUUC50    AUGCCUCACCA61    (2) INFORMATION FOR SEQ ID NO:30:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 61 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    GGCCAAACUGAGCAGACACUAAAUUUGCACGAGGAAGGUUCGCCCCCUUC50    AUGCCUCACCA61    (2) INFORMATION FOR SEQ ID NO:31:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 61 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    GGCCAAACUGAGCAGACCCUAAAUCUGCCCCCGGAAGGUUCGUGCCCUUC50    AUGCCUCACCA61    (2) INFORMATION FOR SEQ ID NO:32:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    GCCAAACTGACGTCATCGACTTCG24    (2) INFORMATION FOR SEQ ID NO:33:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    AACAGCTATGACCATG16    (2) INFORMATION FOR SEQ ID NO:34:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 59 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    GTAATACGACTCACTATAGGCCAAACTGAGCAGACTCTAAATCTGCAAAC50    GGAAGGTTC59    (2) INFORMATION FOR SEQ ID NO:35:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 47 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    ATGGCTTCGTACCCCTGCCATCAACACGCGTCTGCGTTCGACCAGGC47    (2) INFORMATION FOR SEQ ID NO:36:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 71 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    GGTTAACGTCGGACAGACTCTAAATCTGTTGCGGTCTCCGCGCGCAGGTT50    CAAATCCTGCCGCAGACGTTT71    (2) INFORMATION FOR SEQ ID NO:37:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 71 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:    GGTTAACGTCGGACAGACTCTAAATCTGTTGCGGTCTCCGCGCGCAGGTT50    GAAATCCTGCCGCAGACGTTT71    (2) INFORMATION FOR SEQ ID NO:38:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 54 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:    GGTTAACGTCGGTGCGGTCTCCGCGCGCAGGTTCAAATCCTGCCGCAGAC50    GTTT54    (2) INFORMATION FOR SEQ ID NO:39:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 62 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: tRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:    GGCCAAACUGAGCAGACUCUAAAUCUGCAAACGGAAGGUUCGUGCCCUUC50    AUGCCUCACCAU62    (2) INFORMATION FOR SEQ ID NO:40:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 377 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:    GAAGCUGACCAGACAGUCGCCGCUUCGUCGUCGUCCUCUUCGGGGGAGAC50    GGGCGGAGGGGAGGAAAGUCCGGGCUCCAUAGGGCAGGGUGCCAGGUAAC100    GCCUGGGGGGGAAACCCACGACCAGUGCAACAGAGAGCAAACCGCCGAUG150    GCCCGCGCAAGCGGGAUCAGGUAAGGGUGAAAGGGUGCGGUAAGAGCGCA200    CCGCGCGGCUGGUAACAGUCCGUGGCACGGUAAACUCCACCCGGAGCAAG250    GCCAAAUAGGGGUUCAUAAGGUACGGCCCGUACUGAACCCGGGUAGGCUG300    CUUGAGCCAGUGAGCGAUUGCUGGCCUAGAUGAAUGACUGUCCACGACAG350    AACCCGGCUUAUCGGUCAGUUUCACCU377    (2) INFORMATION FOR SEQ ID NO:41:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:    TAATACGACTCACTATAG18    (2) INFORMATION FOR SEQ ID NO:42:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 29 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:    CGCAGACGGTCCTATAGTGAGTCGTATTA29    (2) INFORMATION FOR SEQ ID NO:43:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 63 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:    ACCGCGCAGCCTGGTCGAACGCAGACGCGTGTTGATGGCAGGGGTCTATA50    GTGAGTCGTATTA63    (2) INFORMATION FOR SEQ ID NO:44:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 29 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:    ATGCCTCGGTCCTATAGTGAGTCGTATTA29    (2) INFORMATION FOR SEQ ID NO:45:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 32 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:    GTGGTGTCTGCGTTCGACCAGCTATGACCATG32    (2) INFORMATION FOR SEQ ID NO:46:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:    GTGGTGTCTGCGTTCGACCAGTATGACCATG31    (2) INFORMATION FOR SEQ ID NO:47:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:    GTGGTGTCTGCGTTCGACTATGACCATG28    (2) INFORMATION FOR SEQ ID NO:48:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:    GTGGTGTCTGCGTTCTATGACCATG25    (2) INFORMATION FOR SEQ ID NO:49:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:    GTGGTGTCTGTATGACCATG20    (2) INFORMATION FOR SEQ ID NO:50:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:    GTGGTGAGGCATTTCAGTTATGACCATG28    (2) INFORMATION FOR SEQ ID NO:51:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:    AUGCCUCACCAC12    (2) INFORMATION FOR SEQ ID NO:52:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:    CGCAGACACCAC12    (2) INFORMATION FOR SEQ ID NO:53:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:    GGACCGAGGCAU12    (2) INFORMATION FOR SEQ ID NO:54:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:    GGACCGUCUGCG12    (2) INFORMATION FOR SEQ ID NO:55:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:    GUCUGCGUUCGA12    (2) INFORMATION FOR SEQ ID NO:56:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:    GAGGCAUUUCAGU13    (2) INFORMATION FOR SEQ ID NO:57:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 46 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:    GACCCCTGCCATCAACACGCGTCTGCGTTCGACCAGGCTGCGCGGT46    (2) INFORMATION FOR SEQ ID NO:58:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:    AGCCUGGUCGAACGCAGACACCAAAA26    (2) INFORMATION FOR SEQ ID NO:59:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:    AGCCUGGUCGAACGCAGACACCAC24    (2) INFORMATION FOR SEQ ID NO:60:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:    CUGGUCGAACGCAGACACCAC21    (2) INFORMATION FOR SEQ ID NO:61:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:    GUCGAACGCAGACACCAC18    (2) INFORMATION FOR SEQ ID NO:62:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:    GAACGCAGACACCAC15    (2) INFORMATION FOR SEQ ID NO:63:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:    CAGACACCAC10    (2) INFORMATION FOR SEQ ID NO:64:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:    GUCGAACGCAGACACCAC18    (2) INFORMATION FOR SEQ ID NO:65:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:    CUGGUCGAACGCAGACACCAC21    __________________________________________________________________________

We claim:
 1. An isolated oligonucleotide molecule comprising an externalguide sequence wherein the external guide sequence comprisesarecognition sequence complementary to a targeted sequence in a targetRNA molecule, and an RNase P binding sequence, wherein the RNase Pbinding sequence comprises a nucleotide sequence base pairing withitself to form a structure similar to the T stem and loop of a precursortRNA, wherein the RNase P binding sequence does not form structuresimilar to all or portions of the variable stem, the variable loop, theanticodon stem and the anticodon loop of a precursor tRNA, and whereinthe external guide sequence promotes eukaryotic RNase P catalyticRNA-mediated cleavage of the target RNA molecule.
 2. An isolatedoligonucleotide molecule comprising an external guide sequence whereinthe external guide sequence comprisesa recognition sequencecomplementary to a targeted sequence in a target RNA molecule, and anRNase P binding sequence, wherein the RNase P binding sequence comprisesa nucleotide sequence base pairing with itself to form a structuresimilar to the T stem and loop of a precursor tRNA, wherein the RNase Pbinding sequence does not form structure similar to all or portions ofthe variable stem, the variable loop, the anticodon stem and theanticodon loop of a precursor tRNA, and an RNase P catalytic sequence,wherein the oligonucleotide molecule cleaves the target RNA.
 3. Theoligonucleotide molecule of claim 2 wherein the RNase P catalyticsequence is an H1 sequence or an M1 sequence.
 4. The oligonucleotidemolecule of claim 2 wherein the recognition sequence comprises an Arecognition arm and a D recognition arm, wherein the A recognition armis located at the 3' end of the RNase P binding sequence and the Drecognition arm is located at the 5' end of the RNase P bindingsequence.
 5. The oligonucleotide molecule of claim 4 wherein the RNase Pcatalytic sequence is linked to the 3' end of the A recognition arm. 6.The oligonucleotide molecule of claim 4 wherein the RNase P catalyticsequence is linked to the 5' end of the D recognition arm.
 7. Theoligonucleotide molecule of claim 4 wherein the RNase P binding sequenceconsists ofa nucleotide sequence base pairing with itself to form astructure similar to the T stem and loop of a precursor tRNA.
 8. Theoligonucleotide molecule of claim 2 wherein the RNase P catalyticsequence comprises the sequence of a naturally occurring RNase Pcatalytic RNA.
 9. The oligonucleotide molecule of claim 2 wherein theoligonucleotide molecule is selected byrandomizing a section of thesequence of the starting oligonucleotide molecule; selecting for asubpopulation of the randomized sequences for their ability toefficiently cleave the target RNA; amplifying those sequences cleavingmore efficiently than the starting oligonucleotide molecule; andrepeating the selection and amplification steps.
 10. A composition forpromoting cleavage of a target RNA wherein the composition comprises theoligonucleotide molecule of claim 2 in a pharmaceutically acceptabledelivery system.
 11. The composition of claim 10 wherein thepharmaceutically acceptable delivery system is selected from the groupconsisting of liposomes, virosomes, microspheres and microcapsules. 12.The composition of claim 10 wherein the pharmaceutically acceptabledelivery system is selected from the group consisting of carrierssuitable for topical, subcutaneous, parenteral, and enteraladministration.
 13. A composition for promoting cleavage of a target RNAwherein the composition comprises an engineered expression vectorencoding the oligonucleotide molecule of claim
 2. 14. The composition ofclaim 13 wherein the engineered expression vector is a viral vectorselected from the group consisting of retroviral vectors,adeno-associated viral vectors and Epstein-Barr viral vectors.
 15. Amethod for cleaving a target RNA comprising bringing into contact, underconditions that promote RNase P cleavage, the target RNA and anoligonucleotide molecule comprising an external guide sequence whereinthe external guide sequence comprisesa recognition sequencecomplementary to a targeted sequence in a target RNA molecule, and anRNase P binding sequence, wherein the RNase P binding sequence comprisesa nucleotide sequence base pairing with itself to form a structuresimilar to the T stem and loop of a precursor tRNA, wherein the RNase Pbinding sequence does not form structure similar to all or portions ofthe variable stem, the variable loop, the anticodon stem and theanticodon loop of a precursor tRNA, and wherein the external guidesequence promotes RNase P catalytic RNA-mediated cleavage of the targetRNA molecule.
 16. The method claim 15 wherein the oligonucleotidemolecule further comprisesan RNase P catalytic sequence, wherein theoligonucleotide molecule cleaves the target RNA.
 17. The method of claim16 wherein the step of bringing into contact is accomplished byadministering to a patient or cells from a patient the oligonucleotidemolecule, andwherein the oligonucleotide molecule is in apharmaceutically acceptable delivery system.
 18. A method for selectinga population of RNase P internal guide sequences that cleave a targetRNA with increased efficiency over a starting RNase P internal guidesequence comprisingrandomizing a section of the starting RNase Pinternal guide sequence; selecting for a subpopulation of the randomizedsequences for their ability to efficiently cleave the target RNA;amplifying those sequences cleaving more efficiently than the startingRNase P internal guide sequence; and repeating the selection andamplification steps.
 19. An isolated oligonucleotide molecule comprisingan external guide sequence wherein the external guide sequencecomprisesa recognition sequence complementary to a targeted sequence ina target RNA molecule, and an RNase P binding sequence; wherein theexternal guide sequence promotes RNase P catalytic RNA-mediated cleavageof the target RNA molecule, and wherein at least one nucleotide in theoligonucleotide molecule is selected from the group consisting ofchemically modified nucleotides and chemically unmodifieddeoxyribonucleotides.
 20. The oligonucleotide molecule of claim 19wherein the recognition sequence comprises an A recognition arm and a Drecognition arm, wherein the A recognition arm is located at the 3' endof the external guide sequence and the D recognition arm is located atthe 5' end of the external guide sequence.
 21. The oligonucleotidemolecule of claim 20 whereinthe A recognition arm comprises a nucleotidesequence including at least seven nucleotides complementary to and basepairing with the substrate immediately 3' to a site in the substrate tobe cleaved to form a structure similar to an aminoacyl acceptor stem,the RNase P binding sequence comprises a nucleotide sequence basepairing with itself to form a structure similar to the T stem and loopof a precursor tRNA, and the D recognition arm comprises a nucleotidesequence including at least three nucleotides complementary to and basepairing with the substrate.
 22. The oligonucleotide molecule of claim 21wherein the RNase P binding sequence comprisesa nucleotide sequence basepairing with itself to form a structure similar to the T stem and loopof a precursor tRNA, and a nucleotide sequence forming structure similarto all or portions of the variable stem, the variable loop, theanticodon stem and the anticodon loop of a precursor tRNA.
 23. Acomposition for promoting cleavage of a target RNA molecule wherein thecomposition comprises the oligonucleotide molecule of claim 19 in apharmaceutically acceptable delivery system.
 24. The composition ofclaim 23 wherein the pharmaceutically acceptable delivery system isselected from the group consisting of liposomes, virosomes, microspheresand microcapsules.
 25. The oligonucleotide molecule of claim 19 whereinone or more of the 2' hydroxyl groups of the ribonucleotides arereplaced with a chemical group selected from the group consisting ofhydrogen, an O-alkyl group, an amino group, and fluorine,wherein one ormore of the phosphate linking groups are replaced with a linking groupselected from the group consisting of methyl phosphonate andphosphorothioate, and wherein replacement of one or more of the 2'hydroxyl groups increases resistance of the external guide sequence tonucleases.
 26. The oligonucleotide molecule of claim 19 wherein one ormore of the 2' hydroxyl groups of the ribonucleotides are replaced withhydrogen or a methoxy group; andwherein one or more of the phosphatelinking groups are replaced with phosphorothioate.
 27. Theoligonucleotide molecule of claim 19 further comprisingan RNase Pbinding sequence, wherein the oligonucleotide molecule cleaves thetarget RNA.
 28. An DNA molecule that encodes an RNA molecule comprisingan external guide sequence wherein the external guide sequencecomprisesa recognition sequence complementary to a targeted sequence ina target RNA molecule, and an RNase P binding sequence, wherein theRNase P binding sequence comprises a nucleotide sequence base pairingwith itself to form a structure similar to the T stem and loop of aprecursor tRNA, wherein the RNase P binding sequence does not formstructure similar to all or portions of the variable stem, the variableloop, the anticodon stem and the anticodon loop of a precursor tRNA, andwherein the external guide sequence promotes eukaryotic RNase Pcatalytic RNA-mediated cleavage of the target RNA molecule.
 29. The DNAmolecule of claim 28 wherein the RNA molecule further comprisesan RNaseP catalytic sequence, wherein the RNA molecule cleaves the target RNA.30. An isolated oligonucleotide molecule comprising an external guidesequence wherein the external guide sequence comprisesan RNase P bindingsequence, wherein the RNase P binding sequence comprises a nucleotidesequence base pairing with itself to form a structure similar to the Tstem and loop of a precursor tRNA, wherein the RNase P binding sequencedoes not form structure similar to all or portions of the variable stem,the variable loop, the anticodon stem and the anticodon loop of aprecursor tRNA, and a recognition sequence complementary to a targetedsequence in a target RNA molecule, wherein the recognition sequencecomprises an A recognition arm and a D recognition arm, wherein the Arecognition arm is located at the 3' end of the RNase P binding sequenceand the D recognition arm is located at the 5' end of the RNase Pbinding sequence, and wherein the external guide sequence promoteseukaryotic RNase P catalytic RNA-mediated cleavage of the target RNAmolecule.